Glycolytic metabolism in cultured cells of the nervous system

The transport and metabolism of glucose was examined in monolayers of C-6 glioma cells. 1) Glucose transport appeared to have both a low (Km = 7.74 mM) and a high (Km = 1.16 mM) affinity site in C-6cells; whereas 2-deoxyglucose had only one (Km = 3.7 mM). 2) A large portion of the accumulated glucose was rapidly metabolized to the two glycolytic end products, lactate and pyruvate, and then extruded into the medium. The temperature-dependent efflux of lactate and pyruvate was linear up to 2 hrs with 6 to 10 times more lactate being extruded into the medium than pyruvate. 3) The efflux of lactate and pyruvate increased with increasing extracellular (medium) pH. The presence of 5 percent CO2 not only inhibited the acid efflux but also inhibited the short-term uptake of glucose. The CO2 effect was attributed to a lowering of the medium pH since bicarbonate alone either increased or did not inhibit efflux. 4) Valinomycin increased the levels of cellular lactate but not those of pyruvate by almost three-fold. Lactate efflux was stimulated while that of pyruvate was inhibited. The addition of 5 percent CO2 increased the cellular levels of both lactate and pyruvate, but unlike valinomycin decreased the acid efflux. Idoacetate inhibited the acid efflux by 50 percent suggesting that glycolysis is necessary for efflux.     

Regulation of glucose utilization in chick embryo fibroblasts by bicarbonate ion

The amount of glucose consumed by chick embryo fibroblasts in primary culture is strongly influenced by the presence of bicarbonate ion in the culture medmum. Cells grown on glucose at physiologic concentration (5.5 mm) and in the absence of bicarbonate ion have a reduced rate of glucose utilization when compared to their counterparts cultivated in medium containing the usual 25 mM bicarbonate. The presence or absence of bicarbonate is without effect on chick embryo fibroblast proliferation over a 6-day growth period. Both lactic acid accumulation per mole of glucose consumed and the utilization of glutamine increase as a function of bicarbonate ion in the growth medium.     

Lactate Utilization by Isolated Cells from Early Neonatal Rat Brain

The utilization of lactate, glucose, 3-hydroxybutyrate, and glutamine has been studied in isolated brain cells from early newborn rats. Isolated brain cells actively utilized these substrates, showing saturation at concentrations near physiological levels during the perinatal period. The rate of lactate utilization was 2.5-fold greater than that observed for glucose, 3-hydroxybutyrate, or glutamine, suggesting that lactate is the main metabolic substrate for the brain immediately after birth. The apparent Km for glucose utilization suggested that this process is limited by the activity of hexokinase. However, lactate, 3-hydroxybutyrate, and glutamine utilization seems to be limited by their transport through the plasma membrane. The presence of fatty acid-free bovine serum albumin (BSA) in the incubation medium significantly increased the rate of lipogenesis from lactate or 3-hydroxybutyrate, although this was balanced by the decrease in their rates of oxidation in the same circumstances. BSA did not affect the rate of glucose utilization. The effect of BSA was due not to the removal of free fatty acid, but possibly to the binding of long-chain acyl-CoA, resulting in the disinhibition of acetyl-CoA carboxylase and citrate carrier.     

The Na+-ionophore monensin enhances glucose uptake in mouse thymocytes

Monensin enhanced 2-deoxyglucose uptake and 3-O-methyl glucose transport in mouse thymocytes, but had no effect on L-glucose transport. Cytochalasin B inhibited monensin induced as well as basal glucose uptake. The enhanced 2-deoxyglucose uptake was time and dose-dependent. The increase in the rate of 2-deoxyglucose uptake induced by monensin was more rapid than that of Na+ uptake. Ouabain did not inhibit monensin-enhanced 2-deoxyglucose uptake. Monensin failed to stimulate 2-deoxyglucose uptake at low concentrations of Na+ (13 mM) or K+ (17 mM), higher concentrations of either cation were required for stimulation. Monensin enhanced glucose uptake also in Ca2+-free medium. The data indicate that the stimulation of 2-deoxyglucose uptake by monensin results from activation of carrier-mediated transport.     

Kinetics of 3-O-methyl glucose transport in red blood cells of newborn pigs

The glucose-permeable fetal red cells in the pig are entirely replaced by glucose-impermeable adult red cells within a month after birth. This study investigates the kinetic parameters of the glucose transport mechanism in newborn pig red cells in comparison with immature adult red cells (reticulocytes) as well as the fully matured adult erythrocytes. Influx and efflux of the nonmetabolizable 3-O-methyl glucose (3-O-M-G) in red cells of newborn pigs saturate at high substrate concentrations and exhibit typical Michaelis-Menten kinetics. Km values for efflux are 15.2 and 18.2 mM for 15 and 22 degrees C, respectively. Q10 computed between 10 and 26 degrees is 5.0. The energy of activation for the transport process is 34,000 cal mol-1. The effectiveness of hexoses in competing with 3-O-M-G in efflux is in the following order: D-glucose greater than D-mannose greater than D-fructose greater than D-galactose. Efflux of 3-O-M-G does not increase with 3-O-M-G or D-ribose in the medium and is reduced by 2,4-dinitroflurobenzene (DNFB), p-chloromercuriphenyl sufonic acid (PCMBS), and phloridzin. The reticulocytes are shown to possess a carrier-mediated transport but with a considerably lower transport rate. As the reticulocytes mature into normal red cells, the carrier transport mechanism is lost.     

Support of Xenopus laevis spermatogenesis in vitro by different energy substrates

The support of Xenopus laevis spermatogenesis in vitro by different energy-yielding substrates has been investigated. Isolated spermatogenic cells maintained their levels of adenosine-triphosphate for 24 h in serum-free medium containing only amino acids as energy substrates. DL-Aminocarnitine, an inhibitor of carnitine palmitoyltransferase, reduced cell viability 87% during a 15-h culture in the same medium, indicating that beta oxidation of endogenous fatty acids is a significant source of energy when exogenous substrates are unavailable. Isolated spermatocytes developed into spermatids for 7 days in medium supplemented with either pyruvate, oxaloacetate, or lactate, with maximal survival and development at 0.5 mM pyruvate, 2.0 mM oxaloacetate, and 4.0 mM lactate. Few spermatocytes survived more than 3 days in serum-free medium supplemented with only glucose and amino acids as energy substrates. In contrast, glucose-supplemented medium supported spermatocyte differentiation for 14 days in testis fragment culture and 7 days in spermatocyte-Sertoli cell cocultures due to the excretion of lactate and pyruvate by Xenopus Sertoli cells during culture in glucose-supplemented medium. Glucose also enhanced spermatocyte development in medium containing dialyzed, heat-inactivated fetal calf serum. Spermatogenic cells oxidized glucose to CO2 with C1 oxidized 6- to 7-fold more than C6, suggesting that glucose may be metabolized in the hexose monophosphate shunt. The results are discussed in comparison to energy metabolism in mammalian testes and spermatogenic cells.     

Glucose and amino acid metabolism in an established line of skeletal muscle cells

The suitability of an established myogenic line (L₆) for the study of skeletal muscle intermediary metabolism was investigated. Myoblasts were grown in tissue culture for ten days at which time they had differentiated into multinucleated myotubes. Myotube preparations were then incubated for up to 96 hours in 10 ml of Dulbecco's modified Eagle medium containing 10% fetal calf serum. Glucose was utilized at a nearly linear rate, 3.0 nmol/min/mg protein. Intracellular glucose was detectable throughout the incubation, even when medium glucose was as low as 16 mg%. During the initial 28 hours of incubation, when net lactate production was observed, only 35% of the glucose utilized was converted to lactate. Alanine was produced in parallel to lactate at an average rate of 0.6 nmol/min/mg protein. In concert with active glutamine utilization, high rates of ammoniagenesis were observed as medium glutamine decreased from 3.3 mM to 0.49 mM and medium ammonia increased from 2.3 mM to 6.2 mM, between zero time and 96 hours of incubation, respectively. The cells maintained stable ATP and citrate levels, and physiologic intracellular lactate/pyruvate ratios (10–24) throughout 96 hours of incubation. These results suggest (1) glucose utilization by skeletal muscle in tissue culture is limited by phosphorylation, not transport; (2) as much as 50% of glucose-derived pyruvate enters mitochondrial pathways; (3) glutamine carbon may be utilized simultaneously with glucose consumption and this process accounts for high rates of ammoniagenesis.     

Reversible independent alterations in glucose transport and metabolism in cultured human cells deprived of glucose

We have measured uptake of ³H-hexoses into diploid human cells by exposing them to brief pulses of isotopic sugar during the log-growth, subconfluent-growth, and confluent-growth (contact inhibited) phases of the strain HSWP derived from human skin. ³H-deoxyglucose appears to be taken up three times faster than ³H-glucose. After exposure to ³H-glucose for longer than one minute, the cells excrete ～70% of the isotope into the medium as lactate. If lactate production (and hence excretion) is abolished by treating the cells with iodoacetic acid or dinitrofluorobenzene, neither of which inhibits transport, the uptake of ³H-glucose is found to be in fact somewhat larger than that of ³H-deoxyglucose. If cells are deprived of glucose for 24 hours, apparent uptake of ³H-glucose is enhanced 10-fold or more. This latter increase is accounted for by 2- to 3-fold enhancement of true transport plus retention of > 90% of the radioactivity, since little lactate is formed or excreted in glucose-deprived cells. Deoxyglucose, galactose, or pyruvate when present during glucose deprivation each have quantitatively different effects on the cells' capacity to produce lactate from a short pulse of glucose, but none of them prevents the enhancement of hexose transport. After restoration of 5 mM glucose to starved cells, their metabolism returns to normal (in the sense that ～70% of the glucose taken up in a pulse is again excreted as lactate), with a half-time of 0.5 hour; but the transport of hexoses returns to control levels much more slowly, with a half-time of ～6 hours. The two processes appear to be independently regulated.     

Net movements of monovalent and bivalent cations, and their relation to energy metabolism, in slices of hepatoma 3924A and of a mammary tumour

1. During incubation at 1° in saline medium buffered either with phosphate or bicarbonate, slices of Morris hepatoma 3924A, and of a chemically induced tumour of rat mammary gland, lost K⁺ and gained Na⁺, Ca²⁺ and water.

2. Upon subsequent incubation at 38° in oxygenated medium, these changes were partially reversed. In the hepatoma, the reaccumulation of K⁺ was equally efficient in phosphate or bicarbonate medium, and in the presence and absence of glucose. Ca²⁺ was extruded in bicarbonate, but not in phosphate medium, and its extrusion was reduced in the presence of glucose.

3. When respiration was inhibited in the presence of glucose, K⁺ transport by the hepatoma continued to an extent which varied with the glycolytic activity of the slices, suggesting that the rate of ATP synthesis was a limiting factor under these conditions.

4. In the absence of glucose, the transport of Na⁺ and K⁺ was completely stopped by respiratory inhibition. However, more than 50% of the O₂ uptake had to be inhibited before any effect on transport was observed, suggesting that the rate of synthesis of ATP from endogenous respiration is in excess of that required to maintain transport.

5. Inhibition of transport by ouabain was accompanied by a 30% fall in the rate of endogenous respiration, and by a fall of 33% in the rate of glycolysis in the presence of cyanide plus glucose.

6. Comparison of the minimum rates of respiration and of glycolysis (in the presence of glucose plus cyanide) required to maintain the maximal extent of K⁺ transport in the hepatoma slices, suggests that ATP derived from oxidative phosphorylation or from anaerobic glycolysis is equally efficient as a source of energy for ion transport.     

Cultured trout liver cells: Utilization of substrates and response to hormones

Summary The characterization of a recently established system for the short-term culture of rainbow trout (Oncorhynchus mykiss) liver cells in chemically defined medium has been extended to studies on the metabolic competence of the cells and the characterization of their response to hormones. Three areas of metabolism have been addressed: a) the utilization of the exogenously added substrates fructose, lactate, glucose, dihydroxyacetone, and glycerol for glucose and lactate formation; b) the effects of the pancreatic hormones insulin and glucagon on cellular glucose formation, lactate formation, and fatty acid synthesis; and c) the effects of insulin and dexamethasone on the estradiol-dependent production of vitellogenin. Incubation of trout liver cells with fructose, lactate, glucose, dihydroxyacetone, or glycerol resulted in enhanced rates of cellular glucose and lactate production. Substrate-induced effects usually were more clearly expressed after extended (20 h) than after acute (5 h) culture periods. Addition of the hormones insulin or glucagon caused dose-dependent alterations in the flux of substrates to glucose and lactate. Rates of de novo synthesis of fatty acids from [¹⁴C]acetate were stimulated by insulin and inhibited by glucagon during acute and extended incubation periods. Treatment of liver cells isolated from male trout for 72 h with estradiol induced vitellogenin production and secretion into the medium. However, the addition of insulin or dexamethasone drastically reduced this estrogen-induced vitellogenesis. These results indicate that trout liver cells cultured in defined medium maintain central metabolic pathways, including glycolysis, gluconeogenesis, lipogenesis, and vitellogenesis as well as their responsiveness to various hormones, for at least 72 h. This cell culture system should provide an excellent model to further characterize metabolic processes in fish liver.     

Oxygen consumption of equine articular chondrocytes: Influence of applied oxygen tension and glucose concentration during culture

We investigated the oxygen (O(2)) uptake of equine articular chondrocytes to assess their reactions to anoxia/re-oxygenation. They were cultured under 5% or 21% gas phase O(2) and at glucose concentrations of 0, 1.0 or 4.5g/L in the culture medium (n=3). Afterwards, the O(2) consumption rate of the chondrocytes was monitored (oxymetry) before and after an anoxia period of 25min. The glucose consumption and lactate release were measured at the end of the re-oxygenation period. The chondrocytes showed a minimal O(2) consumption rate, which was hardly changed by anoxia. Independently from the O(2) tension, glucose uptake by the cells was about 30% of the available culture medium glucose, thus higher for cells at 4.5g/L glucose (n=3). Lactate release was also independent from O(2) tension, but lower for cells at 4.5g/L glucose (n=3). Our observations indicated that O(2) consumption by equine chondrocytes was very low despite a functional mitochondrial respiratory chain, and nearly insensitive to anoxia/re-oxygenation. But the chondrocytes metabolism was modified by an excess of O(2) and glucose.     

Evidence for the autocrine inhibition of glycolysis in human fibroblasts

We investigated the mechanism by which glucose refeeding can reverse the enhancement of glycolysis caused by glucose starvation. Human fibroblasts were deprived of glucose for 18 hr and then refed for 1 hr with either (a) medium from sister glucose-starved cultures (controls), (b) fresh, glucose-containing medium (fresh medium), or (c) medium conditioned for 18 hr by glucose-fed cells (conditioned medium). Despite a lower glucose content, conditioned medium was significantly more effective at inhibiting the accumulation of radio-labeled glucose than fresh medium (74 vs. 49% inhibition). The uptake of 2-deoxyglucose was not affected by either medium, indicating that the site of control of glycolysis was distal to glucose transport and phosphorylation. The active principle was heat labile, dialyzable (Mr less than 12,000) and unrelated to the lactate content of conditioned medium. Medium conditioned by cells exposed to 3-O-methylglucose did not inhibit glycolysis in glucose-starved cells even though long-term exposure to this hexose, like glucose, results in the repression of transport.     

Rates of utilization of glucose, glutamine and oleate and formation of end-products by mouse peritoneal macrophages in culture.

1. The metabolism of mouse thioglycollate-elicited peritoneal macrophages was studied in culture for up to 96 h. 2. The rates of glycolysis, lactate formation and glutamine utilization were approximately linear with time for at least 80 h of culture. 3. The rates of glucose and glutamine utilization by cultured macrophages were approx. 500 and 90 nmol/h per mg of protein respectively. This rate of glucose utilization is at least 50% greater than that previously reported for macrophages during 60 min incubation in a shaking flask; and it is now increased by addition of glutamine to the culture medium. The rate of glutamine utilization in culture is similar to that previously reported for macrophages during 60 min incubation. The major end-product of glucose metabolism is lactate, and those of glutamine metabolism are CO2, glutamate, ammonia and alanine. 4. Oleate was utilized by these cells: 14C from [14C]oleate was incorporated into CO2 and cellular lipid. The highest rate of oleate utilization was observed when both glucose and glutamine were present in the culture medium. The presence of oleate in the culture medium did not affect the rates of utilization of either glucose or glutamine. Of the [14C]oleate incorporated into lipid, approx. 80% was incorporated into triacylglycerol and only 18% into phospholipid. 5. The turnover rate for the total ATP content of the macrophage in culture is about 10 times per minute: the value for the perfused isolated maximally working rat heart is 22. This indicates a high metabolic rate for macrophages, and consequently emphasizes the importance of the provision of fuels for their function in an immune response.     

Transport enhancement and reversal: glucose and 3-O-methyl glucose.

In chick embryo fibroblast cultures the 15- to 30-fold enhancement of D-glucose uptake observed when cells are starved of glucose for 24 hours is not duplicated for derivatives of glucose that compete effectively for uptake and have generally been considered to use the same carrier. 2-deoxy-D-glucose, D-mannose, D-galactose and D-glucosamine are derepressed progressively less sharply in that order with glucosamine uptake never more than doubled by starvation. D-glucose at a concentration of 5.5 mM in the 24-hour conditioning medium is a strong "repressor" resulting in low "transport" behavior for each of the five sugars cited. D-glucosamine is equally effective at the same concentration. A 10-fold reduction in the concentration of glucosamine (0.55 mM) allows for the escape from repression of mannose, glucose, and deoxyglucose uptake while the others remain repressed. Mannose uptake escapes as well when the glucose concentration in the "conditioning" medium is similarly reduced. Under certain conditions of starvation and cell density dramatic effects of supplemental stimulation by insulin can be achieved. Insulin withdrawal interrupts the supplemental stimulation process. Cycloheximide, actinomycin D and cordycepin block both non-insulin and insulin-induced derepression. Short exposure (15-30 minutes) of 24-hour starved cells to glucose (5.5 mM) reduces glucose sharply but does not affect 3-O-methyl glucose uptake. If the exposure is to 2-deoxyglucose (5.5 mM) further derepression of glucose uptake results.     

Influence of different ammonium,lactate and glutamine concentrations on CCO cell growth

In this study the effects of ammonium and lactate on a culture of channel catfish ovary (CCO) cells were examined. We also made investigation on the influence of glutamine, since our previous research revealed that this amino acid stimulated CCO cell growth more than glucose in a concentration-dependent manner. The effect of ammonium in cell culture included the considerable decrease in cell growth rate with eventual growth arrest as well as the retardation of glucose consumption. At ammonium concentrations above 2.5 mM, the cells displayed specific morphological changes. The effect of lactate was different to that of ammonium since the cell growth rate was progressively decreasing with the increase of lactate concentration, whereas the glucose consumption rate remained almost unchanged. Besides that, it was found that lactate was steadily eliminated from the culture medium when its initial concentration was relatively high. The influence of glutamine on CCO cell propagation showed that nutrient requirements of this cell line were mainly dependent on glutamine rather than glucose. The increase in glutamine concentration led to the increase in cell growth rate and consequent ammonia accumulation while the glucose utilization and lactate production were reduced. Without glutamine in culture medium cell growth was arrested. However, the lack of glucose reversed the stimulating effect of glutamine by decreasing cell growth rate and affecting amino acid utilization.     

Enhancement of erythropoietin production in recombinant Chinese hamster ovary cells by sodium lactate addition

The stabilization of optimum pH for cells can cause a higher erythropoietin (EPO) production rate and a good growth rate with the prolonged culture span in recombinant Chinese hamster ovary (r-CHO) cells. Our strategy for stabilizing the optimum pH in this study is to reduce the lactate production by adding sodium lactate to a culture medium. When 40 mM sodium lactate was added, a specific growth rate was decreased by approximately 22% as compared with the control culture. However the culture longevity was extended to 187 h, and more than a 2.7-fold increase in a final accumulated EPO concentration was obtained at 40 mM of sodium lactate. On the condition that caused the high production of EPO, a specific glucose consumption rate and lactate production rate decreased by 23.3 and 52%, respectively. Activity of lactate dehydrogenase (LDH) in r-CHO cells increased and catalyzed the oxidation of lactate to pyruvate, together with the reverse reaction, at the addition of 40 mM sodium lactate. The addition of 40 mM sodium lactate caused the positive effects on a cell growth and an EPO production in the absence of carbon dioxide gas as well as in the presence of carbon dioxide gas by reducing the accumulation of lactate.     

Characteristics of CHO-K1 cell culture producing two types of recombinant soluble thrombomodulin

Recombinant CHO-K1 cells, expressing human soluble thrombomodulin, were cultured in a serum-free medium and characteristics of the culture associated with glucose and lactate were investigated. In 3 L fermentor (3LFM) cultures, the cell density was found to have a proportional relationship with the volumetric glucose consumption rate, and the specific glucose consumption rates were constant at about 0.2 mg/(10⁶ cells·d) despite many differences in the culture conditions. Thus, it was concluded that the glucose consumption rate is little influenced by the condition of the cells or the culture conditions, and that the cell density can be estimated by the glucose consumption rate calculated from glucose measurement. Two types of thrombomodulin (rsTMα and rsTMβ) were produced, in which rsTMβ possesses chondroitin-4-sulfate and has greater anticoagulant activities than rsTMα. Therefore, it is important to investigate the rsTMα and rsTMβ production properties, and to determine the optimal culture conditions for high rsTMβ production. The most important factor to increase the production of rsTMβ relative to rsTMα (the β/α ratio) was effective aeration. Moreover, a lower ratio of lactate production/glucose consumption (the L/G ratio) with sufficient oxygen, high glucose concentration, and a longer medium exchange interval contributed to a higher specific rsTMβ production rate. Since there was a linear relationship between the production rate of each type of rsTM and the overall rsTM production rate per liter, it is expected that the rsTMα and rsTMβ production rates may be able to be estimated from the overall rate and the rsTMβ production increased by increasing the overall rsTM production with a lower L/G ratio.     

Transport of 3-O-methyl D-glucose and beta-methyl D-glucoside by rabbit ileum.

The intestinal transport of three actively transported sugars has been studied in order to determine mechanistic features that, (a) can be attributed to stereo-specific affinity and (b) are common. The apparent affinity constants at the brush-border indicate that sugars are selected in the order, beta-methyl glucose greater than D-galactose greater than 3-O-methyl glucose, (the Km values are 1.23, 5.0 and 18.1 mM, respectively.) At low substrate concentrations the Kt values for Na+ activation of sugar entry across the brush-border are: 27, 25, and 140 mequiv. for beta-methyl glucose, galactose and 3-O-methyl glucose, respectively. These kinetic parameters suggest that Na+, water, sugar and membrane-binding groups are all factors which determine selective affinity. In spite of these differences in operational affinity, all three sugars show a reciprocal change in brush-border entry and exit permeability as Ringer (Na) or (sugar) is increased. Estimates of the changes in convective velocity and in the diffusive velocity when the sugar concentration in the Ringer is raised reveal that with all three sugars, the fractional reduction in convective velocity is approximately equal to the (reduction of diffusive velocity)2. This is consistent with the view that the sugars move via pores in the brush-border by convective diffusion. Theophylline reduces the serosal border permeability to beta-methyl glucose and to 3-O-methyl glucose relatively by the same extent and consequently, increase the intracellular accumulation of these sugars. The permeability of the serosal border to beta-methyl glucose entry is lower than permeability of the serosal border to beta-methyl glucose exit, which suggested that beta-methyl glucose may be convected out of the cell across the lateral serosal border.     

Microbial immunomodulators: mannoprotein constituents of<Emphasis Type="Underline">Candida albicans</Emphasis> with immunomodulatory activity in lymphocyte cultures of normal,glioma-bearing and HIV-infected subjects

The culture kinetics of human tumor kidney cells (TCL 598) grown on microcarriers are compared with media initially supplemented with either glucose alone or a mixture of galactose and glucose. Growth rates and maximal cell densities are similar, but cellular death is much slower in galactose than in glucose. Galactose is metabolized at a much slower specific rate than glucose. Cells grown in the galactose medium show a different pattern of lactate and pyruvate metabolism compared to cells grown in the glucose medium. Growth with galactose also favours oxidation of glutamine to alanine.     

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 × 10⁷ 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. This revised version was published online in July 2006 with corrections to the Cover Date.