The Warburg and Crabtree effects: On the origin of cancer cell energy metabolism and of yeast glucose repression

AbstractDuring the last decades a considerable amount of research has been focused on cancer. Recently, tumor cell metabolism has been considered as a possible target for cancer therapy. It is widely accepted that tumors display enhanced glycolytic activity and impaired oxidative phosphorylation (Warburg effect). Therefore, it seems reasonable that disruption of glycolysis might be a promising candidate for specific anti-cancer therapy. Nevertheless, the concept of aerobic glycolysis as the paradigm of tumor cell metabolism has been challenged, as some tumor cells exhibit high rates of oxidative phosphorylation. Mitochondrial physiology in cancer cells is linked to the Warburg effect. Besides, its central role in apoptosis makes this organelle a promising “dual hit target” to selectively eliminate tumor cells. From a metabolic point of view, the fermenting yeast Saccharomyces cerevisiae and tumor cells share several features. In this paper we will review these common metabolic properties as well as the possible origins of the Crabtree and Warburg effects. This article is part of a Special Issue entitled: Bioenergetics of Cancer

Interaction of Mitochondrial and Epigenetic Regulation in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a pathology preceded mainly by cirrhosis of diverse etiology and is associated with uncontrolled dedifferentiation and cell proliferation processes. Many cellular functions are dependent on mitochondrial function, among which we can mention the enzymatic activity of PARP-1 and sirtuin 1, epigenetic regulation of gene expression, apoptosis, and so on. Mitochondrial dysfunction is related to liver diseases including cirrhosis and HCC; the energetic demand is not properly supplied and mitochondrial morphologic changes have been observed, resulting in an altered metabolism. There is a strong relationship between epigenetics and mitochondrion since the first one is dependent on the correct function of the last one. There is an interest to improve or to maintain mitochondrial integrity in order to prevent or reverse HCC; such is the case of IFC-305 that has a beneficial effect on mitochondrial function in a sequential model of cirrhosis-HCC. In this model, IFC-305 downregulates the expression of PCNA, thymidylate synthase, HGF and its receptor c-Met and upregulates the cell cycle inhibitor p27, thereby decreasing cell proliferation. Both effects, improvement of mitochondria function and reduction of tumor proliferation, suggest its use as HCC chemoprevention or as an adjuvant in chemotherapy

Systemorientierte Analysen von hepatischen 13C-Markierungs- und Stoffwechseldynamiken

In this thesis a data-driven model-based approach was undertaken to analyze the liver central metabolism at the systems-level. The work focused on the model-assisted interpretation of metabolome and fluxome data. Experimental observations of stationary and non-stationary 13C-labeling and metabolite data enabled the identification of metabolic fluxes, metabolite dynamics, and patterns of metabolic control by means of transient 13C-flux analysis, dynamic modeling, and metabolic control analysis. The major goals of this contribution were (i) to establish an experimental set-up and a computational framework for acquiring and analyzing transient 13C-mass fraction data in mammalian cells and to use it to identify metabolic fluxes in HepG2 cells, (ii) to apply transient 13C-flux analysis to quantify the effects of a therapeutic dose of the hypolipidemic drug atorvastatin on the cholesterol pathway and central metabolism in primary rat hepatocytes, and (iii) to provide systems-level analyses of the dynamics and control of the central carbon metabolism in hepatoma cells. The experimental observation of the isotopic transient offers the temporal resolution needed to identify intracellular flux maps in mammalian cells from 13C-labeling experiments. To enable the estimation of metabolic fluxes from non-stationary 13C-labeling data in slow- and non-growing mammalian cell batch and fed-batch cultures, reliable means were provided for simultaneous quenching of metabolism and extraction of intracellular intermediates. The developed sampling procedures and chemical analyses were used to quantify metabolite levels and mass fractions in a dynamic labeling experiment with HepG2 cells using 13C-labeled glucose as substrate. In glycolysis and the pentose-phosphate pathway (PPP), isotopic steady state was reached within 30 min, whereas in the tricarboxylic acid (TCA) cycle isotopic steady state was not attained within 120 min. The experimental data were used to estimate the corresponding flux map. In order to identify the flux distribution, a computational framework was developed. The flux estimation was based on a large-scale stoichiometric model, which was used to estimate effluxes into the biomass, and an isotopomer model of glycolysis, the pentose-phosphate pathway, and the tricarboxylic acid cycle. The split ratio between glycolysis and the pentose-phosphate pathway was determined to 57 % and 43 %. It is worth noting that this was the first time metabolic fluxes were estimated in a mammalian system from a transient 13C-labeling experiment. The effects of a therapeutic concentration of the hypolipidemic drug atorvastatin on cholesterol biosynthesis and central metabolism were determined in primary rat hepatocytes using 13C-labeled glutamine and transient 13C flux analysis. Isotopic steady state was observed within 4 h in the central metabolism but not in the cholesterol pathway, regardless of whether the hypolipidemic agent was administered or not. The flux through the cholesterol pathway was found to drop from 0.27 to 0.08 mmol/(lcv h) in response to the administration of the statin. Only minor differences were determined in the central carbon fluxes between cells treated with 50 nM atorvastatin and untreated cells. The flux control coefficient of the HMG-CoA reductase over the cholesterol synthesis flux was determined to 0.46, i.e. cholesterol biosynthesis is not completely controlled by the HMG-CoA reductase. This means that other reaction steps may be also potent targets for lowering blood cholesterol levels. A dynamic liver central carbon metabolism model was developed from metabolite time-series and applied to break down the control hierarchy in hepatoma cells. The dynamic metabolite data were collected from HepG2 cells in a stimulus response experiment in which the cells had been deprived of extracellular glucose. The enzyme kinetics were described with the canonical linlog formalism, which had been reported previously to yield a good approximation quality, while only requiring the determination of comparatively few parameters. The in silico metabolite time-series data were in accordance with the experimentally determined metabolite dynamics. To unravel the internal control structure of the hepatoma central carbon metabolism, concentration and flux control coefficients, partial flux control coefficients, and internal response coefficients were deduced. The control patterns found support the hypotheses that the glucose-6-phosphate dehydrogenase reaction and the Warburg effect (cancer cells have an increased glycolytic flux in the presence of an adequate oxygen supply) are promising targets for tumor treatment.In dieser Arbeit wurde ein datengetriebener modellbasierter Ansatz verfolgt, um den zentralen hepatischen Metabolismus auf Systemebene zu analysieren. Der Schwerpunkt lag dabei auf der modellgestützten Interpretation von Metabolome- und Fluxome-Daten. Die experimentelle Bestimmung von stationären und instationären Markierungs- und Metabolitdaten ermöglichte die Identifizierung von metabolischen Flüssen, Stoffwechsel-Dynamiken und metabolischen Kontrollprinzipien auf Basis von transienter 13C-Stoffflussanalyse, dynamischer Modellierung und metabolischer Kontrollanalyse. Die Hauptziele dieser Arbeite waren (i) die Etablierung eines experimentellen Set-ups und eines rechenbasierten Auswertungsrahmenwerks zur Erhebung und Analyse transienter Massenisotopomerdaten in Säugetierzellen und deren Einsatz zur Identifizierung metabolischer Flüsse in HepG2-Zellen, (ii) die Anwendung der instationären 13C Stoffflussanalyse zur Quantifizierung der Effekte einer therapeutischen Dosis des cholesterinsenkenden Medikaments Atorvastatin auf die Cholesterol-Synthese und den Zentralstoffwechsel in primären Rattenhepatozyten und (iii) die Untersuchung der Dynamik und Kontrolle des zentralen Kohlenstoffwechsels in Hepatoma-Zellen auf Systemebene. Die experimentelle Beobachtung der Markierungsdynamik bietet die erforderliche zeitliche Auflösung, um intrazelluläre Flussverteilungen auch in Säugetierzellen auf Grundlage von Markierungsexperimenten bestimmen zu können. Um die tracerbasierte Schätzung metabolischer Flüsse auch in Batch und Fed-Batch Fermentationen von langsam und nicht wachsenden Säugetierzellen zu ermöglichen, wurden zunächst geeignete Techniken zum gleichzeitigen Quenching des Metabolismus und zur Extraktion intrazellulärer Metabolite etabliert. Die entwickelte Probenahmetechniken und Analytikmethoden wurden dann verwendet, um Metabolitkonzentrationen und Massenfraktionen in HepG2-Zellen in einem dynamischen Markierungsexperiment mit 13C-gelabelter Glucose zu bestimmen. Zur Flussschätzung wurde ein rechenbasiertes Auswertungsrahmenwerk entwickelt. In diesem Rahmenwerk ist es möglich, metabolische Flüsse und Konzentrationen aus stationären und instationären Markierungsdaten zu schätzen. Im Rahmen der Flussschätzung wurde ein großskaliges stöchiometrisches Modell entworfen und dazu verwendet, Abflüsse in die Biomasse zu schätzen. Zur Auswertung der Markierungsdaten wurde ein Isotopomerenmodell der Glykolyse, des Pentosephosphatwegs und des Tricarbonzyklus eingesetzt. Das Verhältnis zwischen dem Fluss in die Glykolyse und in den Pentosephosphatweg wurde zu 57 % und 43 % bestimmt. Basierend auf einem Markierungsversuch mit 13C-gelabeltem Glutamine und instationärer 13C Stoffflussanalyse wurden die Effekte einer therapeutischen Konzentration des Medikaments Atorvastatin auf die Cholesterol-Biosynthese und den Zentrallstoffwechsel von primären Rattenhepatozyten quantifiziert. Unabhängig von der Anwendung des Medikaments stellte sich isotopische Stationarität im Zentralkohlenstoffwechsel innerhalb von 4 h ein, jedoch nicht im Cholesterolstoffwechsel. Als Reaktion auf die Gabe des Medikaments sank der Fluss durch den Cholesterolweg von 0,27 auf 0,08 mmol/(lcv h). Nur unwesentliche Unterschiede wurden im zentralen Kohlenstoffwechsel zwischen Zellen festgestellt, die in 50 nM Atorvastatin kultiviert worden waren, und unbehandelten Zellen. Der Flusskontrollkoeffizient der HMG-CoA Reduktase über den Cholesterol-Synthesefluss wurde zu 0,46 bestimmt, d.h. die Cholesterol-Biosynthese wird nicht ausschließlich durch die HMG-CoA Reduktase kontrolliert. Daraus ergibt sich, dass evtl. auch andere Reaktionsschritte interessante Eingriffsmöglichkeiten zur Senkung der Blutcholesterolwerte bieten. Ein dynamisches Model des zentralen Leberstoffwechsels wurde ausgehend von Metabolitzeitreihen entwickelt und dazu verwendet die Kontrollhierarchie in Hepatoma-Zellen aufzuklären. Die Metabolitdaten wurden in einem Stimulus-Response Experiment mit HepG2-Zellen erhoben, bei dem der Stoffwechsel durch Wegnahme der extrazellulären Glucose ausgelenkt wurde. Die Enzymkinetiken wurden durch den kanonischen Linlog-Ansatz beschrieben, der durch ein gutes Approximationsvermögen bei gleichzeitig verhältnismäßig wenig zu schätzenden Parametern charakterisiert ist. Die in silico Zeitreihen stimmten gut mit den experimentell beobachteten Stoffwechsel-Dynamiken überein. Um die interne Kontrollstruktur des zentralen Kohlenstoffwechsels in Hepatoma-Zellen aufzuklären, wurden Konzentrations- und Flusskontrollkoeffizienten sowie partielle Flusskontrollkoeffizienten und partielle interne Responsekoeffizienten bestimmt. Die gefundenen Kontrollmuster unterstützen Hypothesen nach denen sich die Glucose-6-Phosphate Dehydrogenase und der Warburg-Effekt (Krebszellen haben einen erhöhten glycolytischen Fluss bei ausreichender Sauerstoffversorgung) als Targets zur Tumortherapie eignen

Critical perspective on the consequences of the limited availability of kinetic data in metabolic dynamic modeling

Detailed kinetic models at the network reaction level are usually constructed using enzymatic mechanistic rate equations and the associated kinetic parameters. However, during the cellular life cycle thousands of different reactions occur, which makes it very difficult to build a detailed large-scale ldnetic model. In this work, we provide a critical overview of specific limitations found during the reconstruction of the central carbon metabolism dynamic model from E. coli (based on kinetic data available). In addition, we provide clues that will hopefully allow the systems biology community to more accurately construct metabolic dynamic models in the future. The difficulties faced during the construction of dynamic models are due not only to the lack of kinetic information but also to the fact that some data are still not curated. We hope that in the future, with the standardization of the in vitro enzyme protocols the approximation of in vitro conditions to the in vivo ones, it will be possible to integrate the available kinetic data into a complete large scale model. We also expect that collaborative projects between modellers and biologists will provide valuable kinetic data and permit the exchange of important information to solve most of these issues.Rafael S. Costa would like to thank Fundacao para a Ciencia e Tecnologia for providing the grant SFRH/BD/25506/2005. The authors also acknowledge the MIT-Portugal project 'Bridging Systems and Synthetic Biology for the development of improved microbial cell factories' MIT-Pt/BS-BB/0082/2008

Incorporating genome-scale tools for studying energy homeostasis

Mammals have evolved complex regulatory systems that enable them to maintain energy homeostasis despite constant environmental challenges that limit the availability of energy inputs and their composition. Biological control relies upon intricate systems composed of multiple organs and specialized cell types that regulate energy up-take, storage, and expenditure. Because these systems simultaneously perform diverse functions and are highly integrated, they are extremely difficult to understand in terms of their individual component contributions to energy homeostasis. In order to provide improved treatments and clinical options, it is important to identify the principle genetic and molecular components, as well as the systemic features of regulation. To begin, many of these features can be discovered by integrating experimental technologies with advanced methods of analysis. This review focuses on the analysis of transcriptional data derived from microarrays and how it can complement other experimental techniques to study energy homeostasis

The Impact of Mitochondrial Fission-Stimulated ROS Production on Pro-Apoptotic Chemotherapy.

Cancer is one of the world's deadliest afflictions. Despite recent advances in diagnostic and surgical technologies, as well as improved treatments of some individual tumor types, there is currently no universal cure to prevent or impede the uncontrolled proliferation of malignant cells. Targeting tumors by inducing apoptosis is one of the pillars of cancer treatment. Changes in mitochondrial morphology precede intrinsic apoptosis, but mitochondrial dynamics has only recently been recognized as a viable pharmacological target. In many cancers, oncogenic transformation is accompanied by accumulation of elevated cellular levels of ROS leading to redox imbalance. Hence, a common chemotherapeutic strategy against such tumor types involves deploying pro-oxidant agents to increase ROS levels above an apoptotic death-inducing threshold. The aim of this chapter is to investigate the benefit of stimulating mitochondrial fission-dependent production of ROS for enhanced killing of solid tumors. The main question to be addressed is whether a sudden and abrupt change in mitochondrial shape toward the fragmented phenotype can be pharmacologically harnessed to trigger a burst of mitochondrial ROS sufficient to initiate apoptosis specifically in cancer cells but not in non-transformed healthy tissues

3,5-Diiodo-L-Thyronine Modifies the Lipid Droplet Composition in a Model of Hepatosteatosis

Background/Aims: Fatty acids are the main energy stores and the major membrane components of the cells. In the hepatocyte, fatty acids are esterified to triacylglycerols (TAGs) and stored in lipid droplets (LDs). The lipid lowering action of 3,5-diiodo-L-thyronine (T 2 ) on an in vitro model of hepatosteatosis was investigated in terms of fatty acid and protein content of LDs, lipid oxidation and secretion. Methods: FaO cells were exposed to oleate/ palmitate, then treated with T 2 . Results: T 2 reduced number and size of LDs, and modified their acyl composition by decreasing the content of saturated (SFA) vs monounsaturated (MUFA) fatty acids thus reversing the SFA/MUFA ratio. The expression of the LD-associated proteins adipose differentiation-related protein (ADRP), oxidative tissue-enriched PAT protein (OXPAT), and adipose triglyceride lipase (ATGL) was increased in 'steatotic' cells and further up-regulated by T 2 . Moreover, T 2 stimulated the mitochondrial oxidation by up-regulating carnitine-palmitoyl-transferase (CPT1), uncoupling protein 2 (UCP2) and very long-chain acylcoenzyme A dehydrogenase (VLCAD). Conclusions: T 2 leads to mobilization of TAGs from LDs and stimulates mitochondrial oxidative metabolism of fatty acids, in particular of SFAs, and thus enriches of MUFAs the LDs. This action may protect the hepatocyte from excess of SFAs that are more toxic than MUFAs

Biological Activities of Some Algal Extracts from Lebanon; Antioxidant, Anti-inflammatory, and Anti-steatotic Effects

Nonalcoholic fatty liver disease (NAFLD) is a metabolic manifestation of liver disease, characterized by lipid accumulation in hepatocytes. Despite starting as a benign disorder, NAFLD can progress to more severe pathologies such as nonalcoholic steatohepatitis, fibrosis, cirrhosis, and eventually hepatocellular carcinoma. The epidemiological prevalence of this disease is 25 % of the general population, and its worsening suggests that NAFLD is supposed to be the main cause for liver transplantation. NAFLD pharmacological treatment is still limited by unwanted side effects, whereas the best therapeutical approach is a lifestyle that depends on physical exercises and a healthy diet. The use of natural-derived compounds with therapeutic potential is advisable. Recently, medicinal plants applications have been focused on renewable sources as marine algae, especially seaweeds. Seaweeds are characterized with huge amounts of phytochemicals such as fucoidan (FUC) polysaccharide, which is known for its antioxidant, anti-inflammatory, anti-cancer, and hepatoprotective effects. In the present work, we extracted a water soluble fucoidan fraction from the brown algae Cystoseira compressa, and for the first time from the roots of the terrestrial shrub Ferula hermonis. These fucoidan fraction were termed CYS and FER, respectively. Then together with a previously purified fucoidan from the terrestrial plant Eucalyptus globulus \u201cEUC\u201d were studied and compared for their chemical features and biological activities. CYS, FER, and EUC contained fucose, glucose, sulfate, smaller amounts of monosaccharides such as galactose and mannose, and a minor quantity of proteins. FUCs structural features were investigated by FTIR, 1H NMR and 13C NMR spectroscopy. The antioxidant properties of FUCs were measured by DPPH, ABTS and FRAP assays, results revealed a high radical scavenging capacity that was confirmed in in vitro cellular models. In hepatic and endothelial cells, FUC reduced ROS production induced by intracellular lipid accumulation. Moreover, in our NAFLD model that consists of hepatic and endothelial cells treated with a fatty acid mixture prior to the FUCs treatment, FUCs purified from the three vegetal species exhibited a significant antisteatotic action; being able to reduce intracellular triglyceride content and to regulate the expression of key genes of hepatic lipid metabolism. Altogether, our results candidate CYS, FER, and EUC as possible bioactive compounds against fatty liver disease and related vascular damage. Results indicated that among the purified FUCs, that purified from the terrestrial plant E. globulus was the most biologically active extract