Mammalian Cell Metabolism Research Articles

Metabolic-flux analysis of continuously cultured hybridoma cells using (13)CO(2) mass spectrometry in combination with (13)C-lactate nuclear magnetic resonance spectroscopy and metabolite balancing.

Protein production of mammalian-cell culture is limited due to accumulation of waste products such as lactate, CO(2), and ammonia. In this study, the intracellular fluxes of hybridoma cells are measured to determine the amount by which various metabolic pathways contribute to the secretion of waste products derived from glucose. Continuously cultured hybridoma cells are grown in medium containing either 1-(13)C-, 2-(13)C-, or 6-(13)C-glucose. The uptake and production rates of amino acids, glucose, ammonia, O(2), and CO(2) as well as the cellular composition are measured. In addition, the (13)C distribution of the lactate produced and alanine produced by the hybridomas is determined by (1)H-NMR spectroscopy, and the (13)CO(2)/(12)CO(2) ratio is measured by on-line mass spectrometry. These data are used to calculate the intracellular fluxes of the glycolysis, the pentose phosphate pathway, the TCA cycle, and fluxes involved in amino acid metabolism. It is shown that: (i) approximately 20% of the glucose consumed is channeled through the pentose shunt; (ii) the glycolysis pathway contributes the most to lactate production, and most of the CO(2) is produced by the TCA cycle; (iii) the pyruvate-carboxylase flux is negligibly small; and (iv) the malic-enzyme flux is estimated to be 10% of the glucose uptake rate. Based on these flux data suggestions are made to engineer a more efficient glucose metabolism in mammalian cells.

S-Nitrosylation Is Emerging as a Specific and Fundamental Posttranslational Protein Modification: Head-to-Head Comparison with O-Phosphorylation

Nitric oxide (NO) is a free-radical product of mammalian cell metabolism that plays diverse and important roles in the regulation of cell function. Biological actions of NO arise as a direct consequence of chemical reactions between NO or NO-derived species and protein targets. Reactions of NO with transition metals in target proteins have garnered the most attention to date as the principal mechanism of NO signaling; nonetheless, S-nitrosylation of protein Cys residues is rapidly moving to center stage in importance. In general, however, there has been a delay in adequate appreciation of the role of S-nitrosylation in biological signaling by NO. This lag is attributed to a poor understanding of the basis for selective targeting of NO to particular thiols, and methodological limitations in accurately quantifying this modification--recent breakthroughs in concepts and methods diminish these barriers. Here, we consider the wheres and whys of protein S-nitrosylation and its basis for specificity. Protein S-nitrosylation potentially represents a ubiquitous and fundamental mechanism for posttranslational control of protein activity on a par with that of O-phosphorylation.

Fluid shear stress increases membrane fluidity in endothelial cells: a study with DCVJ fluorescence.

Fluid shear stress (FSS) has been shown to be an ubiquitous stimulator of mammalian cell metabolism. Although many of the intracellular signal transduction pathways have been characterized, the primary mechanoreceptor for FSS remains unknown. One hypothesis is that the cytoplasmic membrane acts as the receptor for FSS, leading to increased membrane fluidity, which in turn leads to the activation of heterotrimetric G proteins (13). 9-(Dicyanovinyl)-julolidine (DCVJ) is a fluorescent probe that integrates into the cell membrane and changes its quantum yield with the viscosity of the environment. In a parallel-plate flow chamber, confluent layers of DCVJ-labeled human endothelial cells were exposed to different levels of FSS. With increased FSS, a reduced fluorescence intensity was observed, indicating an increase of membrane fluidity. Step changes of FSS caused an approximately linear drop of fluorescence within 5 s, showing fast and almost full recovery after shear cessation. A linear dose-response relationship between shear stress and membrane fluidity changes was observed. The average fluidity increase over the entire cell monolayer was 22% at 26 dyn/cm(2). This study provides evidence for a link between FSS and membrane fluidity, and suggests that the membrane is an important flow mechanosensor of the cell.

Material balance studies on animal cell metabolism using a stoichiometrically based reaction network

A detailed reaction network of mammalian cell metabolism contains hundreds of enzymatic reactions. By grouping serial reactions into single overall reactions and separating overlapped pathways into independent reactions, the total number of reactions of the network is significantly reduced. This strategy of manipulating the reaction network avoids the manipulations of a large number of reactions otherwise needed to determine the reaction extents. A stoichiometric material balance model is developed based on the stoichiometry of the simplified reaction network. Closures of material balances on glucose and each of the 20 amino acids are achieved using experimental data from three controlled fed-batch and one-batch hybridoma cultures. Results show that the critical role of essential amino acids, except glutamine, is to provide precursors for protein synthesis. The catabolism of some of the essential amino acids, particularly isoleucine and leucine, is observed when an excess amount of these amino acids is available in the culture medium. It was found that the reduction of glutamine utilization (for reducing ammonia production) is accompanied by an increase in the uptake of nonessential amino acids (NAAs) from the culture medium. This suggests that NAAs are necessary even though they are not essential for cell growth. A glutamine balance shows that less than 20% of the glutamine nitrogen is utilized for essential roles, such as protein and nucleotide syntheses. A relatively constant percentage (about 45%) of the glutamine nitrogen is utilized for NAA biosynthesis, despite the fact that the absolute amount varies among the four experiments. As to the carbon skeleton of glutamine, a significant portion enters the tricarboxylic acid (TCA) cycle. A material balance on glucose shows that most of the glucose (81%) is converted into lactate when glucose is in excess. On the other hand, when glucose is limited, lactate production is considerably reduced, while a major portion of glucose (48%) enters the TCA cycle. The fraction of glucose used for the synthesis of cellular components ranges from 9 to 28%.

A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: Effect of nutrient concentration, dilution rate, and pH

Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutaminE, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.

The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells

The control of RNA synthesis from protein-coding genes is fundamental in determining the various cell types of higher eukaryotes. The activation of these genes is driven by promoter complexes, and RNA synthesis is performed by an enzyme mega-complex-the RNA polymerase II holoenzyme. These two complexes are the fundamental components required to initiate gene expression and generate the primary transcripts that, after processing, yield mRNAs that pass to the cytoplasm where protein synthesis occurs. But although this gene expression pathway has been studied intensively, aspects of RNA metabolism remain difficult to comprehend. In particular, it is unclear why >95% of RNA polymerized by polymerase II remains in the nucleus, where it is recycled. To explain this apparent paradox, this review presents a detailed description of nuclear RNA (nRNA) metabolism in mammalian cells. We evaluate the number of active transcription units, discuss the distribution of polymerases on active genes, and assess the efficiency with which the products mature and pass to the cytoplasm. Differences between the behavior of mRNAs on this productive pathway and primary transcripts that never leave the nucleus lead us to propose that these represent distinct populations. We discuss possible roles for nonproductive RNAs and present a model to describe the metabolism of these RNAs in the nuclei of mammalian cells.-Jackson, D. A., Pombo, A., Iborra, F. The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells.

Cross-talk of NO, superoxide and molecular oxygen, a majesty of aerobic life

Because nitric oxide (NO) reacts with various molecules, such as hemeproteins, superoxide and thiols including glutathione (GSH) and cysteine residues in proteins, biological effects and metabolic fate of this gaseous radical are affected by these reactants. Although the lifetime of NO is short particularly under air atmospheric conditions (where the oxygen tension is unphysiologically high), it increases significantly under physiologically low oxygen concentrations. Because oxygen tensions in human body differ from one tissue to another and change depending on their metabolism, biological activity of NO in various tissues might be affected by local oxygen tensions. To elucidate the role of NO and related radicals in the regulation of circulation and energy metabolism, their effects on arterial resistance and energy metabolism in mitochondria, mammalian cells and enteric bacteria were studied under different oxygen tensions. Kinetic analysis revealed that NO-dependent generation of cGMP in resistance arteries and their relaxation were strongly enhanced by lowering oxygen tensions in the medium. NO reversibly suppressed the respiration and ATP synthesis of isolated mitochondria and intact cells particularly under low oxygen tensions. Kinetic analysis revealed that cross-talk between NO and superoxide generated in and around endothelial cells regulates arterial resistance particularly under physiologically low oxygen tensions. NO also inhibited the respiration and ATP synthesis of E. coli particularly under low oxygen tensions. Because concentrations of NO and H+ in gastric juice are high, most ingested bacteria are effectively killed in the stomach. However, the inhibitory effects of NO on the respiration and ATP synthesis of H. pylori are extremely small. Kinetic analysis revealed that H. pylori generates the superoxide radical thereby inhibiting the bactericidal action of NO in gastric juice. Based on such observations, critical roles of the cross-talk of NO, superoxide and molecular oxygen in the regulation of energy metabolism and survival of aerobic and microaerophilic organisms are discussed.

The role of reactive oxygen species in atherosclerosis.

Reactive oxygen species (ROS) are probably not only unintended, toxic side-products of oxygen metabolism in mammalian cells, they also have several important physiologic functions including antimicrobial killing, regulation of cellular proliferation and growth, and regulation of vascular tone. ROS are generated within the vessel wall by several mechanisms, including a vascular type of a NAD(P)H oxidase. ROS formation can be stimulated by mechanical stress, environmental factors, the peptide angiotensin II, cytokines, native low-density lipoproteins (LDL), and in the presence of catalytic metal ions. Their ability to modify LDL, react with endothelial-derived nitric oxide subsequently forming peroxynitrite, and amplify the expression of various genes important for leukocyte recruitment within the arterial wall are the basis of the oxidant injury theory of atherosclerosis. In animal studies, antioxidant therapy (probucol, butylated hydroxytoluene, N', N'-diphenylenediamide, vitamin E, superoxide dismutase) have been successfully used to prevent fatty streak formation, and to restore impaired nitric oxide-dependent vasorelaxation. In man, antioxidant therapy (e.g., supplementation with vitamin E) clearly increased the resistance of LDL to oxidative modification. Case-controlled and prospective clinical studies suggest a relation between baseline antioxidant plasma levels and/or antioxidant supplementation and risk of cardiovascular events. In one secondary prevention trial (randomized, blinded, placebo-controlled), vitamin E supplementation reduced significantly the risk for non-fatal myocardial infarctions. Before general recommendations can be made, results of further large-scale trials should be awaited.

Fluxes and enzyme activities in central metabolism of myeloma cells grown in chemostat culture.

Activities of enzymes in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and glutaminolysis have been determined in the mouse myeloma SP2/0.Ag14. Cells were grown on IMDM medium with 5% serum in steady-state chemostat culture at a fixed dilution rate of 0.03 h-1. Three culture conditions, which differed in supply of glucose and oxygen, were chosen so as to change catabolic fluxes in the central metabolism, while keeping anabolic fluxes constant. In the three steady-state situations, the ratio between specific rates of glucose and glutamine consumption differed by more than twentyfold. The specific rates of glucose consumption and lactate production were highest at low oxygen supply, whereas the specific rate of glutamine consumption was highest in the culture fed with low amounts of glucose. Under low oxygen conditions, the specific production of ammonia increased and the consumption pattern of amino acids showed large changes compared with the other two cultures. For the three steady states, activities of key enzymes in glycolysis, the pentose phosphate pathway, glutaminolysis, and the TCA cycle were measured. The differences in the in vivo fluxes were only partially reflected in changes in enzyme levels. The largest differences were observed in the levels of glycolytic enzymes, which were elevated under conditions of low oxygen supply. High activities of phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32) in all cultures suggest an important role for this enzyme as a link between glutaminolysis and glycolysis. For all enzymes, in vitro activities were found that could accommodate the estimated maximum in vivo fluxes. These results show that the regulation of fluxes in central metabolism of mammalian cells occurs mainly through modulation of enzyme activity and, to a much lesser extent, by enzyme synthesis.

Translocation of PDK-1 to the plasma membrane is important in allowing PDK-1 to activate protein kinase B.

Protein kinase B (PKB) is involved in the regulation of apoptosis, protein synthesis and glycogen metabolism in mammalian cells. Phosphoinositide-dependent protein kinase (PDK-1) activates PKB in a manner dependent on phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), which is also needed for the translocation of PKB to the plasma membrane. It has been proposed that the amount of PKB activated is determined exclusively as a result of its translocation, and that a constitutively active pool of membrane-associated PDK-1 simply phosphorylates all the PKB made available. Here, we have investigated the effects of membrane localisation of PDK-1 on PKB activation. Ectopically expressed PDK-1 translocated to the plasma membrane in response to platelet-derived growth factor (PDGF) and translocation was sensitive to wortmannin, an inhibitor of phosphoinositide 3-kinase. Translocation of PDK-1 also occurred upon its co-expression with constitutively active phosphoinositide 3-kinase, but not with an inactive form. Overexpression of PDK-1 enhanced the ability of PDGF to activate PKB. PDK-1 disrupted in the pleckstrin homology (PH) domain which did not translocate to the membrane did not increase PKB activity in response to PDGF, whereas membrane-targeted PDK-1 activated PKB to the extent that it could not be activated further by PDGF. In response to PDGF, binding of Ptdlns (3,4,5)P3 and/or Ptdlns(3,4)P2 to the PH domain of PDK-1 causes its translocation to the plasma membrane where it co-localises with PKB, significantly contributing to the scale of PKB activation.

Converse Modulation of IRP1 and IRP2 by Immunological Stimuli in Murine RAW 264.7 Macrophages

Iron regulatory proteins (IRP1 and IRP2) are two cytoplasmic RNA-binding proteins that control iron metabolism in mammalian cells. Both IRPs bind to specific sequences called iron-responsive elements (IREs) located in the 3' or 5' untranslated regions of several mRNAs, in particular mRNA encoding ferritin and transferrin receptor. In this study, we followed in parallel the in vivo regulation of the two IRPs in physiologically stimulated macrophages. We show that stimulation of mouse RAW 264.7 macrophage-like cells increased IRP1 IRE binding activity 4-fold, whereas IRP2 activity decreased 2-fold 8 h after interferon-gamma/lipopolysaccharide treatment. Decrease in IRP2 was not due to nitric oxide (NO) production and did not require de novo protein synthesis. Our data therefore indicate that the two IRPs can be conversely regulated in response to the same stimulus. In addition, the effect of endogenously produced NO on IRP1 was further characterized in an activated macrophage/target cell system. We show that NO acts as an intercellular signal to increase IRP1 activity in adjacent cells. As the effect was detectable within 1 h and did not require de novo protein synthesis, this result supports a direct action of NO on IRP1.

N-Acylethanolamines: formation and molecular composition of a new class of plant lipids

Recently, the biosynthesis of an unusual membrane phospholipid, N-acylphosphatidylethanolamine (NAPE), was found to increase in elicitor-treated tobacco (Nicotiana tabacum L.) cells (K.D. Chapman, A. Conyers-Hackson, R.A. Moreau, S. Tripathy [1995] Physiol Plant 95: 120-126). Here we report that before induction of NAPE biosynthesis, N-acylethanolamine (NAE) is released from NAPE in cultured tobacco cells 10 min after treatment with the fungal elicitor xylanase. In radiolabeling experiments [14C]NAE (labeled on the ethanolamine carbons) increased approximately 6-fold in the culture medium, whereas [14C]NAPE associated with cells decreased approximately 5-fold. Two predominant NAE molecular species, N-lauroylethanolamine and N-myristoylethanolamine, were specifically identified by gas chromatography-mass spectrometry in lipids extracted from culture medium, and both increased in concentration after elicitor treatment. NAEs were found to accumulate extracellularly only. A microsomal phospholipase D activity was discovered that formed NAE from NAPE; its activity in vitro was stimulated about 20-fold by mastoparan, suggesting that NAPE hydrolysis is highly regulated, perhaps by G-proteins. Furthermore, an NAE amidohydrolase activity that catalyzed the hydrolysis of NAE in vitro was detected in homogenates of tobacco cells. Collectively, these results characterize structurally a new class of plant lipids and identify the enzymatic machinery involved in its formation and inactivation in elicitor-treated tobacco cells. Recent evidence indicating a signaling role for NAPE metabolism in mammalian cells (H.H.O. Schmid, P.C. Schmid, V. Natarajan [1996] Chem Phys Lipids 80: 133-142) raises the possibility that a similar mechanism may operate in plant cells.

Probing the metabolism of genetically-engineered mammalian cells by heat flux

With the onset of the commercial production of target proteins by hybridoma and genetically engineered cells, there is a pressing requirement for biosensors to monitor on-line and in real-time their growth in culture. In terms of irreversible thermodynamics, most of the Gibbs energy provided in substrates for this process is dissipated as heat, with only a relatively small quantity being conserved in biomass and the target proteins. Calorimetry would appear, therefore, to be a strong contender for a metabolic probe. A flow microcalorimeter was modified for use with animal cells and tested using CHO 320 cells which produce the heterologous glycoprotein dimer, Interferon-γ. Preliminary results showed that heat flow did not follow the increase in cell-number concentration and it was realised that this technique must be combined with one to assess biomass on-line accurately to reflect metabolic activity. Dielectric spectroscopy was chosen because capacitance measurements of the culture measures the volume fraction of viable cells and not the dead ones. A commercial version, the viable cell monitor, was validated by parallel measurements of the volume of viable cells by flow cytometry. The combined probe showed that heat flux was a function of the specific growth rate which, in turn, was related to the fluxes for glucose and glutamine utilisation and possibly to the accumulation of toxic end products, lactate and ammonia. The calorimetric-respirometric (CR) ratio was highly negative during growth in the fully aerobic conditions, indicating simultaneous anaerobic metabolism during growth. The CR ratio gave a value consistent with solely oxidative processes once there was no growth. This might indicate that ATP demand was greater than could be satisfied by oxidative processes, but this was not the case since the cells were respiring at only ca. 60% of capacity, as judged by uncoupling with FCCP. It appears likely that lactate production was due to the need to produce biosynthetic precursors which were not supplied in the medium. A better medium design would probably decrease this requirement and thereby reduce the accumulation of toxic end products. The heat flux probe was shown to be invaluable in exploring the metabolism of CHO 320 cells grown in batch culture.

Isolation and Characterization of a Prenylcysteine Lyase from Bovine Brain

Prenylated proteins contain one of two isoprenoid lipids, either the 15-carbon farnesyl or the 20-carbon geranylgeranyl, covalently attached to cysteine residues at or near their C terminus. The cellular abundance of prenylated proteins, which can comprise up to 2% of total cellular protein, raises the question of how cells dispose of prenylcysteines produced during the normal turnover of prenylated proteins. We have identified and characterized a novel enzyme, which we term prenylcysteine lyase, that is capable of cleaving the thioether bond of prenylcysteines. The enzyme was isolated from bovine brain membranes and exhibits an apparent molecular mass of 63 kDa. The enzyme did not require NADPH as cofactor for prenylcysteine degradation, thus distinguishing it from cytochrome P450- and flavin-containing monooxygenases that catalyze S-oxidation of thioethers. Purified prenylcysteine lyase shows similar kinetics in utilization of both farnesylcysteine and geranylgeranylcysteine as substrates, although Vmax is 2-fold higher with the former compound. Interaction of prenylcysteine substrates with the enzyme requires that they possess a free amino group; N-acetylated prenylcysteines and prenyl peptides are not substrates. These findings suggest that prenylcysteine lyase is a specific enzyme involved in prenylcysteine metabolism in mammalian cells, most likely comprising the final step in the degradation of prenylated proteins.

Studies of metabolic control using NMR and molecular genetics

The techniques of NMR spectroscopy and molecular genetics have provided new and powerful approaches to studying the control and organisation of cellular metabolism in vivo. We review here our recent applications of these methodologies to the study of energy metabolism in yeast and mammalian cells.

Glycosylation of CHO-derived recombinant tPA produced under elevated pCO2.

Carbon dioxide is a metabolic byproduct of mammalian cell metabolism that can accumulate in poorly ventilated cultures. A buildup of CO2 at constant pH will be accompanied by an increase in medium osmolality. We have examined the glycosylation of tissue plasminogen activator (tPA) produced under serum-free conditions by recombinant Chinese hamster ovary (CHO) cells (MT2-1-8 cell line) in response to elevated pCO2 at constant or elevated osmolality. The proportion of sialic acids comprising N-glycolylneuraminic acid decreased from 2.3-4.0% under 36 mmHg pCO2 to 1.5-2.2% under 250 mmHg pCO2. No changes were observed in the total sialic acid content, the content of other monosaccharides, the relative amounts of type I and type II tPAs, the distribution of surface charges, or the proportion of high-mannose oligosaccharides-even though these conditions have previously been shown to inhibit the specific growth rate of MT2-1-8 cells by 30-40% and the specific tPA production rate by as much as 40%. These results suggest robust glycosylation of tPA by CHO cells.

Effects of glutamine supply on growth and metabolism of mammalian cells in chemostat culture

Glutamine is a major source of energy, carbon, and nitrogen for mammalian cells. The amount of glutamine present in commercial mammalian cell media is, however, not necessarily balanced with cell requirements. Therefore, the effects of glutamine limitation on the physiology of two mammalian cell lines were studied in steady-state chemostat cultures fed with IMDM medium with 5% serum. The cell lines used were MN12, a mouse-mouse hybridoma, and SP2/0-Ag14, a mouse myeloma often used in hybridoma fusions. Cultures, grown at a fixed dilution rate of 0.03 h(-1), were fed with media containing glutamine concentrations ranging from 0.5 to 4 mmol L(-1). Biomass dry weight and cell number were linearly proportional to the glutamine concentrations fed, between 0.5 and 2 mmol L(-1), and glutamine was completely consumed by both cell lines. From this it was concluded that glutamine was the growth-limiting substrate in this concentration range and that the standard formulation of IMDM medium contains a twofold excess of glutamine. In glutamine-limited cultures, the specific rates of ammonia and alanine production were low compared to glutamine-excess cultures containing 4 mmol L(-1) glutamine in the feed medium. The specific consumption rates of nearly all amino acids decreased with increasing glutamine feed, indicating that, in their metabolic function, they may partially be replaced by glutamine. Both cell lines reacted similarly to differences in glutamine feeding in all aspects investigated, except for glucose metabolism, In SP2/0-Ag14 glutamine feed concentrations did not affect the specific glucose consumption, whereas in MN12 this parameter increased with increasing amounts of glutamine fed. This systematic study using controlled culture conditions together with a detailed analysis of culture data shows that, although cells may react similarly in many aspects, cell-line-specific characteristics may be encountered even with respect to fundamental physiological responses like the interaction of the glutamine and glucose metabolism.

Effects of glutamine supply on growth and metabolism of mammalian cells in chemostat culture

Effects of glutamine supply on growth and metabolism of mammalian cells in chemostat culture

Model Applicable to NMR Studies for Calculating Flux Rates in Five Cycles Involved in Glutamate Metabolism

Based on the same principles as those utilized in a recent study for modeling glucose metabolism (Martin, G., Chauvin, M. F., Dugelay, S., and Baverel, G. (1994) J. Biol. Chem. 269, 26034-26039), a method is presented for determining metabolic fluxes involved in glutamate metabolism in mammalian cells. This model consists of five different cycles that operate simultaneously. It includes not only the tricarboxylic acid cycle, the "oxaloacetate --> phosphoenolpyruvate --> pyruvate --> oxaloacetate" cycle and the "oxaloacetate --> phosphoenolpyruvate --> pyruvate --> acetyl-CoA --> citrate --> oxaloacetate" cycle but also the "glutamate --> alpha-ketoglutarate --> glutamate" and the "glutamate --> glutamine --> glutamate" cycles. The fates of each carbon of glutamate, expressed as ratios of integrated transfer of this carbon to corresponding carbons in subsequent metabolites, are described by a set of equations. Since the data introduced in the model are micrograms of atom of traced carbon incorporated into each carbon of end products, the calculation strategy was determined on the basis of the most reliable parameters determined experimentally. This model, whose calculation routes offer a large degree of flexibility, is applicable to data obtained by 13C NMR spectroscopy, gas chromatography - mass spectrometry, or 14C counting in a great variety of mammalian cells.

Alteration of mammalian cell metabolism by dynamic nutrient feeding.

The metabolism of hybridoma cells was controlled to reduce metabolic formation in fed-batch cultures by dynamically feeding a salt-free nutrient concentrate. For this purpose, on-line oxygen uptake rate (OUR) measurement was used to estimate the metabolic demand of hybridoma cells and to determine the feeding rate of a concentrated solution of salt-free DMEM/F12 medium supplemented with other medium components. The ratios among glucose, glutamine and other medium components in the feeding nutrient concentrate were adjusted stoichiometrically to provide balanced nutrient conditions for cell growth. Through on-line control of the feeding rate of the nutrient concentrate, both glucose and glutamine concentrations were maintained at low levels of 0.5 and 0.2 mM respectively during the growth stage. The concentrations of the other essential amino acids were also maintained without large fluctuations. The cell metabolism was altered from that observed in batch cultures resulting in a significant reduction of lactate, ammonia and alanine production. Compared to a previously reported fed-batch culture in which only glucose was maintained at a low level and only a reduced lactate production was observed, this culture has also reduced the production of other metabolites, such as ammonium and alanine. As a result, a high viable cell concentration of more than 1.0 × 10(7) cells/mL was achieved and sustained over an extended period. The results demonstrate an efficient nutrient feeding strategy for controlling cell metabolism to achieve and sustain a high viable cell concentration in fed-batch mammalian cell cultures in order to enhance the productivity.