Metabolic Plasticity in Resting and Thrombin Activated Platelets

Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand.     

Comparative analysis of glucose and glutamine metabolism in transformed mammalian cell lines,insect and primary liver cells

Glucose and glutamine metabolism in several cultured mammalian cell lines (BHK, CHO, and hybridoma cell lines) were investigated by correlating specific utilization and formation rates with specific maximum activities of regulatory enzymes involved in glycolysis and glutaminolysis. Results were compared with data from two insect cell lines and primary liver cells. Flux distribution was measured in a representative mammalian (BHK) and an insect (Spodoptera frugiperda) cell line using radioactive substrates. A high degree of similarity in many aspects of glucose and glutamine metabolism was observed among the cultured mammalian cell lines examined. Specific glucose utilization rates were always close to specific hexokinase activities, indicating that formation of glucose-6-phosphate from glucose (catalyzed by hexokinase) is the rate limiting step of glycolysis. No activity of the key enzymes connecting glycolysis with the tricarboxylic acid cycle, such as pyruvate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase, could be detected. Flux distribution in BHK cells showed glycolytic rates very similar to lactate formation rates. No glucose- or pyruvate-derived carbon entered the tricarboxylic acid cycle, indicating that glucose is mainly metabolized via glycolysis and lactate formation. About 8% of utilized glucose was metabolized via the pentose phosphate shunt, while 20 to 30% of utilized glucose followed pathways other than glycolysis, the tricarboxylic acid cycle, or the pentose phosphate shunt. About 18% of utilized glutamine was oxidized, consistent with the notion that glutamine is the major energy source for mammalian cell lines. Mammalian cells cultured in serum-free low-protein medium showed higher utilization rates, flux rates, and enzyme activities than the same cells cultured in serum-supplemented medium. Insect cells oxidized glucose and pyruvate in addition to glutamine. Furthermore, insect cells produced little or no lactate and were able to channel glycolytic intermediates into the tricarboxylic acid cycle. Metabolic profiles of the type presented here for a variety of cell lines may eventually enable one to interfere with the metabolic patterns of cells relevant to biotechnology, with the hope of improving growth rate and/or productivity. © 1996 Wiley-Liss, Inc.     

Glutamine and glucose as energy substrates for Ehrlich ascites tumour cells

Energy metabolism of freshly harvested Ehrlich ascites tumour cells in the presence of 5 mM glucose and/or 0.5 mM glutamine was studied. The rate of oxygen utilization was not altered by the addition of 0.5 mM glutamine; 5 mM glucose induced an inhibition of respiration. In the presence of both glucose and glutamine, the Crabtree effect decreased. In these conditions, the rates of oxygen uptake, the CO2 evolution and the changes in the redox states of cytochromes indicate that glucose is preferred by Ehrlich ascites tumour cells as energy substrate. Glucose decreased the rate of glutamine utilization by 34%. On the other hand, glutaminolysis did not inhibit glycolysis.     

Heterogeneity in tumor cell energetic metabolome at different cell cycle phases of human colon cancer cell lines

In this present study, the efficacy of metabolomics as a tool for tumor cell energetics for in vitro cell cultures was demonstrated with full competence for the first time by elucidating the anabolic and energy-yielding segments of glycolysis and glutaminolysis, which constitute a part of energy metabolism in tumor cells. By synchronizing colon cancer cells SW480 and SW620 in culture, the metabolome specific to cell cycle phases was analyzed using nuclear magnetic resonance spectroscopy. At the G₁/S transition of the cell cycle (i.e. transition from cell growth to duplication of genetic material), the majority of the energy production was realized by glycolysis through a high channeling of glucose carbons towards lactate. During the late S phase, the majority of energy was produced by glutaminolysis through a high channeling of glutamine carbons towards lactate, while the glucose carbons were channeled towards bio-synthetic pathways. These results indicate that the metabolism of proliferating cells is heterogeneous throughout the cell cycle and can be better interpreted on the basis of different cell cycle phases. These findings could be exploited for the development of a tool for tumor diagnosis as well as for targeting tumors.     

Metabolism of radiolabelled energy-yielding substrates by rat Sertoli cells

The rates of metabolism in vitro of 3H- or 14C-labelled glucose, pyruvate, glutamine and leucine by Sertoli cells from immature rats were estimated. The overall rate of glucose utilization exceeded by far the rates of oxidation of pyruvate (derived from glucose) via the citric acid cycle and glucose metabolism via the oxidative branch of the pentose phosphate pathway. This pattern of glucose metabolism was not markedly altered after stimulation of glucose metabolism by FSH. The rate of oxidation of exogenous pyruvate indicated that the energy yield from glucose metabolism by Sertoli cells could be dependent on the extracellular concentrations of pyruvate and lactate. There is no evidence that a high rate of aerobic glycolysis is of vital importance for Sertoli cells. In medium containing glucose and all amino acids, 14C-labelled glutamine and leucine were converted to 14CO2 at considerable rates. It was calculated that the oxidation of glutamine and leucine in addition to glucose and fatty acids can yield much of the required energy of Sertoli cells.     

Kinetic characterization of vero cell metabolism in a serum‐free batch culture process

A global kinetic study of the central metabolism of Vero cells cultivated in a serum‐free medium is proposed in the present work. Central metabolism including glycolysis, glutaminolysis, and tricarboxylic acid cycle (TCA) was demonstrated to be saturated by high flow rates of consumption of the two major substrates, glucose, and glutamine. Saturation was reavealed by an accumulation of metabolic intermediates and amino acids, by a high production of lactate needed to balance the redox pathway, and by a low participation of the carbon flow to the TCA cycle supply. Different culture conditions were set up to reduce the central metabolism saturation and to better balance the metabolic flow rates between lactate production and energetic pathways. From these culture conditions, substitutions of glutamine by other carbon sources, which have lower transport rates such as asparagine, or pyruvate in order to shunt the glycolysis pathway, were successful to better balance the central metabolism. As a result, an increase of the cell growth with a concomitant decrease of cell death and a better distribution of the carbon flow between TCA cycle and lactate production occurred. We also demonstrated that glutamine was a major carbon source to supply the TCA cycle in Vero cells and that a reduction of lactate production did not necessary improve the efficiency of the Vero cell metabolism. Thus, to adapt the formulation of the medium to the Vero cell needs, it is important to provide carbon substrates inducing a regulated supply of carbon in the TCA cycle either through the glycolysis or through other pathways such as glutaminolysis. Finally, this study allowed to better understand the Vero cell behavior in serum‐free medium which is a valuable help for the implementation of this cell line in serum‐free industrial production processes. Biotechnol. Bioeng. 2010;107: 143–153. © 2010 Wiley Periodicals, Inc.     

Molecular intricacies of aerobic glycolysis in cancer: current insights into the classic metabolic phenotype

Aerobic glycolysis is the process of oxidation of glucose into pyruvate followed by lactate production under normoxic condition. Distinctive from its anaerobic counterpart (i.e. glycolysis that occurs under hypoxia), aerobic glycolysis is frequently witnessed in cancers, popularly known as the “Warburg effect”, and it is one of the earliest known evidences of metabolic alteration in neoplasms. Intracellularly, aerobic glycolysis circumvents mitochondrial oxidative phosphorylation (OxPhos), facilitating an increased rate of glucose hydrolysis. This in turn enables cancer cells to successfully compete with normal cells for glucose uptake in order to maintain uninterrupted growth. In addition, evading OxPhos mitigates excessive generation/accumulation of reactive oxygen species that otherwise may be deleterious to cells. Emerging data indicate that aerobic glycolysis in cancer also promotes glutaminolysis to satisfy the precursor requirements of certain biosynthetic processes (e.g. nucleic acids). Next, the metabolic intermediates of aerobic glycolysis also feed the pentose phosphate pathway (PPP) to facilitate macromolecular biosynthesis necessary for cancer cell growth and proliferation. Extracellularly, the extrusion of the end-product of aerobic glycolysis, i.e. lactate, alters the tumor microenvironment, and impacts cancer-associated cells. Collectively, accumulating data unequivocally demonstrate that aerobic glycolysis implicates myriad of molecular and functional processes to support cancer progression. This review, in the light of recent research, dissects the molecular intricacies of its regulation, and also deliberates the emerging paradigms to target aerobic glycolysis in cancer therapy.     

Bonded Cumomer Analysis of Human Melanoma Metabolism Monitored by 13C NMR Spectroscopy of Perfused Tumor Cells

A network model for the determination of tumor metabolic fluxes from ¹³C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mm [1,6-¹³C₂]glucose under normoxic conditions at 37 °C and monitored by ¹³C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was ∼50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was ∼6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mm) and euglycemic (5 mm) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism.     

Glutaminolysis and glycolysis regulation by troglitazone in breast cancer cells: Relationship to mitochondrial membrane potential

We studied the roles of glycolysis and glutaminolysis following an acute reduction in mitochondrial membrane potential (Ψ_m) induced by the thiazolidinedione troglitazone (TRO) and compared the responses with CCCP‐induced depolarization in breast cancer derived MCF‐7 and MDA‐MB‐231 cells as well as in the MCF‐10A normal breast cell line. TRO and CCCP both acutely reduced Ψ_m but after 24 h TRO‐treated cells had restored Ψ_m associated with both increased glycolysis and glutaminolysis. In contrast, CCCP‐treated cells exhibited only a partial restoration of Ψ_m associated with increased glycolysis but decreased glutaminolysis. TRO‐induced glutaminolysis was coupled to increased ammonium (GDH flux) and decreased alanine production (ALT flux) in all three cell lines. Both cancer cell lines exhibited a higher spontaneous GDH/ALT flux than the normal breast cell line associated with a reduced keto‐acid pool. TRO's effect on GDH/ALT fluxes and mitochondrial keto‐acid pool homeostasis was additive with glucose withdrawal suggesting limited intramitochondrial pyruvate availability. The TRO‐induced acceleration in GDH flux supplies substrate for Complex I contributing to the restoration of Ψ_m as well as Krebs cycle intermediates for biosynthesis. Inhibiting mitochondrial proton ATPase with oligomycin or nullifying the proton gradient with CCCP prevented both the TRO‐induced recovery of Ψ_m and accelerated GDH flux but restored ALT flux consonant with important roles for proton pumping in regulating GDH flux and Ψ_m recovery. Blocking enhanced GDH flux reduced DNA synthesis consistent with glutaminolysis via GDH playing an important biosynthetic role in tumorigenesis. J. Cell. Physiol. 226: 511–519, 2011. © 2010 Wiley‐Liss, Inc.     

Maximum activities of some key enzymes of glycolysis, glutaminolysis, Krebs cycle and fatty acid utilization in bovine pulmonary endothelial cells

Despite the importance of endothelial cells little is known about their metabolic fuel requirements. To provide some information in this area, the maximum catalytic activities of key enzymes of important metabolic pathways have been measured in bovine pulmonary endothelial cells. The results suggest that both glucose and glutamine are important fuels for these cells: in addition, the oxidation of fatty acids may also be of quantitative significance. The activity of glutaminase in these cells was about 20-fold higher than that in lymphocyte, a cell which exhibits high rates of glutaminolysis. It is suggested that a high rate of glutamine metabolism by endothelial cells is important not only for energy provision but also for provision of nitrogen for biosynthetic purposes including production of local messengers.     

Glutaminolysis: Supplying carbon or nitrogen or both for cancer cells?

A cancer cell comprising largely of carbon, hydrogen, oxygen, phosphorus, nitrogen and sulfur requires not only glucose, which is avidly transported and converted to lactate by aerobic glycolysis or the Warburg effect, but also glutamine as a major substrate. Glutamine and essential amino acids, such as methionine, provide energy through the TCA cycle as well as nitrogen, sulfur and carbon skeletons for growing and proliferating cancer cells. The interplay between utilization of glutamine and glucose is likely to depend on the genetic make-up of a cancer cell. While the MYC oncogene induces both aerobic glycolysis and glutaminolysis, activated b-catenin induces glutamine synthesis in hepatocellular carcinoma. Cancer cells that have elevated glutamine synthetase can use glutamate and ammonia to synthesize glutamine and are hence not addicted to glutamine. As such, cancer cells have many degrees of freedom for re-programming cell metabolism, which with better understanding will result in novel therapeutic approaches.     

Metabolic reprogramming in cancer: unraveling the role of glutamine in tumorigenesis

Increased glutaminolysis is now recognized as a key feature of the metabolic profile of cancer cells, along with increased aerobic glycolysis (the Warburg effect). In this review, we discuss the roles of glutamine in contributing to the core metabolism of proliferating cells by supporting energy production and biosynthesis. We address how oncogenes and tumor suppressors regulate glutamine metabolism and how cells coordinate glucose and glutamine as nutrient sources. Finally, we highlight the novel therapeutic and imaging applications that are emerging as a result of our improved understanding of the role of glutamine metabolism in cancer.     

The role of glutamine and glucose analogues in metabolic inhibition of human myeloid leukaemia In vitro

• 1.1.Glutamine analogues l-[alphaS,5S]-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) and 6-diazo-5-oxo-l-norleucine (DON) have been shown to possess cytotoxic activity against a wide variety of animal and human xenografted solid tumours, however their potential in man has been limited by toxicity.
• 2.2.We have analysed the effects of acivicin and DON on glutamine utilization to determine whether the reason for the disappointing therapeutic profile is solely due to the inefficient inhibition of glutamine metabolism.
• 3.3.Human myeloid leukaemic cells treated with acivicin inhibited ribonucleotide biosynthesis but not energy production via glutaminolysis and had little effect on viability, whereas treatment with DON inhibited both ribonucleotide biosynthesis and glutamine oxidation and resulted in reduced viability.
• 4.4.Treatment of the myeloid leukaemic cells with the glucose analogue 2-deoxy-d-glucose in addition to DON potentiated the inhibition of de novo nucleotide biosynthesis, glutaminolysis and glycolysis, and caused a further reduction in cell viability.
• 5.5.These results provide further support for the essential role of glutamine in cellular metabolism, and indicate that use of the glutamine analogue DON in the treatment of acute myeloid leukaemia may be more clinically effective if used in combination with 2-deoxy-d-glucose.

     

Lactate promotes glutamine uptake and metabolism in oxidative cancer cells

Oxygenated cancer cells have a high metabolic plasticity as they can use glucose, glutamine and lactate as main substrates to support their bioenergetic and biosynthetic activities. Metabolic optimization requires integration. While glycolysis and glutaminolysis can cooperate to support cellular proliferation, oxidative lactate metabolism opposes glycolysis in oxidative cancer cells engaged in a symbiotic relation with their hypoxic/glycolytic neighbors. However, little is known concerning the relationship between oxidative lactate metabolism and glutamine metabolism. Using SiHa and HeLa human cancer cells, this study reports that intracellular lactate signaling promotes glutamine uptake and metabolism in oxidative cancer cells. It depends on the uptake of extracellular lactate by monocarboxylate transporter 1 (MCT1). Lactate first stabilizes hypoxia-inducible factor-2α (HIF-2α), and HIF-2α then transactivates c-Myc in a pathway that mimics a response to hypoxia. Consequently, lactate-induced c-Myc activation triggers the expression of glutamine transporter ASCT2 and of glutaminase 1 (GLS1), resulting in improved glutamine uptake and catabolism. Elucidation of this metabolic dependence could be of therapeutic interest. First, inhibitors of lactate uptake targeting MCT1 are currently entering clinical trials. They have the potential to indirectly repress glutaminolysis. Second, in oxidative cancer cells, resistance to glutaminolysis inhibition could arise from compensation by oxidative lactate metabolism and increased lactate signaling.     

In vitro effect of extracellular AMP on MCF-7 breast cancer cells: inhibition of glycolysis and cell proliferation.

MCF-7 human breast cancer cells propagated in vitro were treated with adenosine derivatives added to the culture medium. The effects on cell proliferation, glycolysis, and glutaminolysis were investigated. Of all adenosine derivatives tested, AMP was the most efficient inhibitor of cell proliferation. In AMP-treated cells, DNA synthesis decreased, whereas RNA and protein syntheses rose normally with time. In terms of carbohydrate metabolism, lactate production from glucose was drastically reduced; therefore, most of lactate produced must have been derived from glutamine. Increases in the enzyme activities involved in glutamate degradation and in the malate-aspartate shuttle were observed. In contrast, actual glycolytic flux rates declined, whereas key glycolytic enzyme activities increased. Metabolites such as fructose 1,6-bisphosphate and pyruvate accumulated in AMP-arrested cells. Based on the lowered NAD level in the AMP-treated cells, lactate dehydrogenase, but not malate dehydrogenase, was impaired; thereby the whole of glycolysis was inhibited. In compensation, glutamine catabolism was increased. NAD concentrations fell drastically because of the known inhibition of P-ribose-PP synthesis through heightened intracellular AMP levels. A hypothetical metabolic scheme to explain these results and to show how extracellular AMP may influence carbohydrate metabolism and cell proliferation is presented.     

Effect of hypo- and hyperthyroidism on the function and metabolism of macrophages in rats

The effect of thyroid hormones on monocyte migration, phagocytic capacity and hydrogen peroxide production by macrophages and the effect of these hormones on glutamine and glucose metabolism was investigated. The experiments were performed on resident, thioglycollate- and BCG-stimulated cells from hypo- and hyperthyroid rats. High plasma levels of thyroid hormones suppressed the migration of monocytes and hydrogen peroxide production, whereas hypothyuroidism did not affect cell migration but rasied the phagocytic capacity and the hydrogen peroxide production. Hyperthyroidism increased the activities of glutaminase and hexokinase and the rates of decarboxylation of [U-¹⁴C]-glutamine and [U-¹⁴C]-glucose in inflammatory and activated cells. Hypothyroidism stimulated glucose metabolism and had only a slight effect on glutaminolysis. The activity of the TCA cycle was, however, diminished in the presence of high plasma levels of thyroid hormones and enhanced by the hypothyroid state. These findings suggest that the functional changes observed are more likely to be related to the activity of the TCA cycle rather than to glutaminolysis and glycolysis.     

Malate-citrate cycle during glycolysis and glutaminolysis in Ehrlich ascites tumor cells

The malate-citrate cycle was studied during aerobic glycolysis and glutaminolysis in a strain of Ehrlich ascites tumor cells which showed a very low malate-aspartate shuttle system activity. The experimental approach includes: estimation of mitochondrial NAD[P]+-dependent malic enzyme activity; respiratory activity of freshly harvested or fasted cells, and of isolated mitochondria; and determination of the metabolites involved in the glycolytic and glutaminolytic pathways. The results suggest that in this strain, the malate-citrate shuttle is not an effective pathway for transferring glycolytic reducing equivalents from cytosol to mitochondria. Less than 15% of the glucose uptake was affected by the 1,2,3-benzenetricarboxylate inhibition of the malate-citrate shuttle. Moreover, in the presence of glucose, the malate-citrate cycle did not appear to play an important role in the glutaminolytic process. The present work supports and extends the finding of previous studies, since the results showed that the glucose metabolism depressed the oxidative processes in Ehrlich ascites tumor mitochondria, not only alone, but also in the presence of glutamine. Interestingly, the high glutamine uptake was maintained in the presence of glucose.     

Effects of glucose and insulin on HepG2‐C3A cell metabolism

HepG2, hepatocellular carcinoma cells, are used in drug toxicity studies and have also been explored for bioartificial livers. For these applications, the cells are under variable levels of nutrients and hormones, the effects of which on metabolism are poorly understood. In this study, HepG2‐C3A cells were cultured under varying levels of glucose (high, low, and glucose‐free) and insulin (without and with physiological levels of insulin) for 5 days. Cell growth was found to be comparable between high and low glucose media and lowest for glucose‐free medium. Several features of central metabolism were affected profoundly by the medium glucose levels. Glucose consumption was greater for low glucose medium compared to high glucose medium, consistent with known glucose feedback regulation mechanisms. Urea productivity was highest in glucose‐free medium. Further, it was seen that lactate acted as an alternative carbon source in the absence of glucose, whereas it acted as a sink for the high and low glucose media. Using a metabolic network flexibility analysis (MNFA) framework with stoichiometric and thermodynamic constraints, intracellular fluxes under varying levels of glucose and insulin were evaluated. The analysis indicates that urea production in HepG2‐C3A cells arises via the arginase II pathway rather than from ammonia detoxification. Further, involvement of the putrescine metabolism with glutamine metabolism caused higher urea production in glucose‐free medium consistent with higher glutamine uptake. MNFA indicated that in high and low glucose media, glycolysis, glutaminolysis, and oxidative phosphorylation were the main sources of energy (NADH, NADPH, and ATP). In the glucose‐free medium, due to very low glycolytic flux, higher malate to pyruvate glutaminolytic flux and TCA cycle contributed more significantly to energy metabolism. The presence of insulin lowered glycerol uptake and corresponding fluxes involved in lipid metabolism for all glucose levels but otherwise exerted negligible effect on metabolism. HepG2‐C3A cells thus show distinct differences from primary hepatocytes in terms of energy metabolism and urea production. This knowledge can be used to design media supplements and metabolically engineer cells to restore necessary hepatic functions to HepG2‐C3A cells for a range of applications. Biotechnol. Bioeng. 2010;107: 347–356. © 2010 Wiley Periodicals, Inc.     

Glycolysis is important for optimal asexual growth and formation of mature tissue cysts by Toxoplasma gondii

Toxoplasma gondii can grow and replicate using either glucose or glutamine as the major carbon source. Here, we have studied the essentiality of glycolysis in the tachyzoite and bradyzoite stages of T. gondii, using transgenic parasites that lack a functional hexokinase gene (Δhk) in RH (Type-1) and Prugniaud (Type-II) strain parasites. Tachyzoite stage Δhk parasites exhibit a fitness defect similar to that reported previously for the major glucose transporter mutant, and remain virulent in mice. However, although Prugniaud strain Δhk tachyzoites were capable of transforming into bradyzoites in vitro, they were severely compromised in their ability to make mature bradyzoite cysts in the brain tissue of mice. Isotopic labelling studies reveal that glucose-deprived tacyzoites utilise glutamine to replenish glycolytic and pentose phosphate pathway intermediates via gluconeogenesis. Interestingly, while glutamine-deprived intracellular Δhk tachyzoites continued to replicate, extracellular parasites were unable to efficiently invade host cells. Further, studies on mutant tachyzoites lacking a functional phosphoenolpyruvate carboxykinase (Δpepck1) revealed that glutaminolysis is the sole source of gluconeogenic flux in glucose-deprived parasites. In addition, glutaminolysis is essential for sustaining oxidative phosphorylation in Δhk parasites, while wild type (wt) and Δpepck1 parasites can obtain ATP from either glycolysis or oxidative phosphorylation. This study provides insights into the role of nutrient metabolism during asexual propagation and development of T. gondii, and validates the versatile nature of central carbon and energy metabolism in this parasite.     

Glycolysis Is Governed by Growth Regime and Simple Enzyme Regulation in Adherent MDCK Cells

Due to its vital importance in the supply of cellular pathways with energy and precursors, glycolysis has been studied for several decades regarding its capacity and regulation. For a systems-level understanding of the Madin-Darby canine kidney (MDCK) cell metabolism, we couple a segregated cell growth model published earlier with a structured model of glycolysis, which is based on relatively simple kinetics for enzymatic reactions of glycolysis, to explain the pathway dynamics under various cultivation conditions. The structured model takes into account in vitro enzyme activities, and links glycolysis with pentose phosphate pathway and glycogenesis. Using a single parameterization, metabolite pool dynamics during cell cultivation, glucose limitation and glucose pulse experiments can be consistently reproduced by considering the cultivation history of the cells. Growth phase-dependent glucose uptake together with cell-specific volume changes generate high intracellular metabolite pools and flux rates to satisfy the cellular demand during growth. Under glucose limitation, the coordinated control of glycolytic enzymes re-adjusts the glycolytic flux to prevent the depletion of glycolytic intermediates. Finally, the model''s predictive power supports the design of more efficient bioprocesses.