Metabolic pathways in Ehrlich ascites tumor cells recovering from a low bioenergetic status

Ehrlich ascites tumor cells were found to be in a low bioenergetic status, as evaluated by acridine orange uptake and ATP content, when resuspended in a glucose medium shortly after removal from the animal. Dye uptake as well as ATP content then increased for about 2 h at room temperature. This effect was only slightly inhibited by oligomycin. Cells resuspended in a glucose-free medium initially showed high dye uptake and ATP level, which were stable over time: in this case oligomycin caused a drop in both dye uptake and ATP level. The above findings, which are indicative of a marked Crabtree effect in Ehrlich ascites tumor cells, means that it is unlikely that limiting ADP and P_i play an important role in the glucose-induced inhibition of oxidative phosphorylation in this system.     

Ammonium ions enhance the flow through the pyruvate dehydrogenase in Ehrlich ascites tumor cells

The flow through pyruvate dehydrogenase was assayed in glycolysing cells by the evolution of 14CO2 from [1-14C] pyruvate. Parallel incubations were carried out in high bicarbonate buffer (25 mM) and in bicarbonate-free buffer. The activation of the complex by NH+4 was only observed in high bicarbonate buffer, because the dilution of labelled CO2 in the presence of an excess of bicarbonate enables the quantitative determination of labelled CO2 evolved from pyruvate in the decarboxylase step. In the bicarbonate-free buffer the activation of the complex was not observed, because the 14CO2 evolved from pyruvate was consumed by biosynthetic processes inside the cell. On the contrary in isolated hepatocytes the NH+4 activation of the pyruvate dehydrogenase was observed in both buffers. In Ehrlich ascites cells, in common with other mammalian tissues, pyruvate dehydrogenase activity was found to be inversely correlated to the intramitochondrial ATP/ADP ratio.     

Cyanide-resistant respiration is frequent, but confined to yeasts incapable of aerobic fermentation

In Pichia membranifaciens, cyanide-resistant respiration (CRR) sensitive to salicylhydroxamic acid emerged after forced aeration of starved cells for 4 h. Surveying a large number of species by this simple methodology, we found that CRR is very frequent among yeasts. Remarkably, considering our results together with previous data in the literature, CRR was present in 24 out of 28 non-fermentative or Crabtree-negative yeasts and absent in 10 out of 12 Crabtree-positive yeasts. We submit that, as alternatives to cytochromic respiration, yeasts developed two strategies: either aerobic fermentation in Crabtree-positive yeasts or CRR in non-fermentative or Crabtree-negative yeasts.     

Regulation of cellular energy metabolism. The Crabtree effect

The Crabtree effect (inhibition of respiration by glycolysis) is observed in cells with approximately equal glycolytic and respiratory capacities for ATP synthesis. Addition of glucose to aerobic suspensions of glucose-starved cells (Sarcoma 180 ascites tumor cells) causes a burst of respiration and lactate production due to ATP utilization for glucose phosphorylation by hexokinase and phosphofructokinase. This burst of activity is followed by inhibition of both respiration and glycolysis, the former to below the value before glucose addition (Crabtree effect). Both the respiratory rate and the glycolytic flux appear to be regulated by the cytosolic $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ albeit by completely different mechanisms. Respiration is regulated by the free energy of hydrolysis of ATP, such that the rate increases as the $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ decreases and decreases as the $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ increases. The regulatory enzymes of glycolysis are activated by ADP (AMP) and P_i and inhibited by ATP. Thus both respiration and glycolysis increase or decrease as the $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ decreases or increases. The parallel regulation of both ATP-producing pathways by this common metabolite ratio is consistent with the cytoplasmic $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ being an important determinant of homeostatic regulation of cellular energy metabolism.     

Occurrence of the Crabtree effect in HeLa cells

The occurrence of a Crabtree effect in HeLa cells was detected. Some properties of pyruvate kinase (PK) were also evaluated. Hexose phosphate, triose-phosphate and phosphoenolpyruvate (PEP) significantly decreased the oxygen consumption of digitonin-permeabilized HeLa cells, which were oxidizing succinate. The Crabtree effect promoted by PEP was concentration-dependent and was lowered by an increase of ADP concentration, suggesting a participation of PK. The dependence of fructose-1,6-bisphosphate (FDP) by HeLa cell PK was observed. The PK of HeLa cells was inhibited by L -alanine only in the absence of FDP, while in the presence of the metabolite, an increase in the activity was observed. PK was also inhibited in the presence of L -histidine and L -leucine, while L -serine promoted activation. L -Cysteine and L -phenylalanine also inhibited the PK of HeLa cells. This, together with the sigmoidal character in relation to substrate concentration, suggests the presence of the K-type of PK in HeLa cells. © 1998 John Wiley & Sons, Ltd.     

Glucose and phosphate inhibit respiration and oxidative metabolism in cultured hamster eight-cell embryos: evidence for the "crabtree effect"

The development of hamster eight-cell embryos is inhibited by glucose in culture medium containing inorganic phosphate (Pi). This is hypothetically attributed to the "Crabtree effect," in which enhanced glycolysis inhibits respiratory activity and oxidative metabolism. To examine this hypothesis, oxygen consumption of hamster eight-cell embryos was measured using a microelectrode. A two- to three-fold decrease in oxygen consumption was observed in embryos cultured with glucose and Pi. Oxidizable substrates and intermediates of the Krebs cycle supported embryo development only in the absence of glucose and Pi; Krebs cycle inhibitors (fluoroacetate and arsenite) arrested embryo development. Under anaerobic conditions, pyruvate and lactate did not support embryo development. Inhibition of both respiration and oxidative activity in cultured hamster embryos by glucose and Pi is consistent with the existence of a Crabtree effect and indicates that the metabolic properties of preimplantation embryonic cells differ markedly from those of most somatic cells and resemble some cancer cells.     

Respirofermentative metabolism in Kluyveromyces lactis: Ethanol production and the Crabtree effect

Kluyveromyces lactis is a yeast widely used in processes related to milk whey use and lactose fermentation. However, contradictory information about some aspects related to the respirofermentative metabolism of this yeast is found in the literature. We have studied ethanol production and oxygen use in discontinuous and continuous cultures of K. lactis under hypoxic and aerobic conditions. Growth in nonfermentable carbon sources reflects a more efficient respiratory capacity of K. lactis in relation to Saccharomyces cerevisiae; however, in both species, similar glucose fermentation levels under aerobic oxygen-limited conditions are found. Continuous K. lactis cultures in fully oxidative conditions show the oxygen and substrate uptake rates typical of a respiration-unlimited Crabtree-negative yeast; however, a small residual fermentation is present even when respiration is not limited. Some aspects of the Crabtree effect in K. lactis are discussed. The impossibility of including K. lactis in any group of the metabolism-based classification from Alexander and Jeffries (1990) has led us to the formulation of a new group which incorporates the peculiarities of this and other related yeasts.     

Oscillatory metabolism of Saccharomyces cerevisiae in continuous culture

Short-period (40-50 min) synchronized metabolic oscillation was found in a continuous culture of yeast Saccharomyces cerevisiae under aerobic conditions at low-dilution rates. During oscillation, many parameters changed cyclically, such as dissolved oxygen concentration, respiration rate, ethanol and acetate concentrations in the culture, glycogen, ATP, NADH, pyruvate and acetate concentrations in the cells. These changes were considered to be associated with glycogen metabolism. When glycogen was degraded, the respiro-fermentative phase was observed, in which ethanol was produced and the respiration rate decreased. In this phase, the levels of intracellular pyruvate and acetate became minimum, ATP became high and intracellular pH at its lowest level. When glycogen metabolism changed from degradation to accumulation, the respiratory phase started, during which ethanol was re-assimilated from the culture and the respiration rate increased. Intracellular pyruvate and acetate became maximum, ATP decreased and the intracellular pH appeared high. These findings may indicate new aspects of the control mechanism of glycogen metabolism and how respiration and ethanol fermentation are regulated together under aerobic conditions.     

NADH reoxidation does not control glycolytic flux during exposure of respiring Saccharomyces cerevisiae cultures to glucose excess

Introduction of the Lactobacillus casei lactate dehydrogenase (LDH) gene into Saccharomyces cerevisiae under the control of the TPI1 promoter yielded high LDH levels in batch and chemostat cultures. LDH expression did not affect the dilution rate above which respiro-fermentative metabolism occurred (Dc) in aerobic, glucose-limited chemostats. Above Dc, the LDH-expressing strain produced both ethanol and lactate, but its overall fermentation rate was the same as in wild-type cultures. Exposure of respiring, LDH-expressing cultures to glucose excess triggered simultaneous ethanol and lactate production. However, the specific glucose consumption rate was not affected, indicating that NADH reoxidation does not control glycolytic flux under these conditions.     

Glucose metabolism and cell size in continuous cultures of Saccharomyces cerevisiae

A detailed analysis of the cell size, monitored as protein content, has been performed in glucose-limited continuous cultures, so as to obtain the values of the average protein content for various subpopulations at different cell cycle stages, as a function of the growth rate. Glucose metabolism appears to affect cell size, since there is an increase of the average protein content of the population when cells produce ethanol above the critical dilution rate. If the production of ethanol is forced at low growth rates by the addition of formate, the average protein content increases. These results indicate a link between glucose metabolism and cell size in budding yeast, as observed for mammalian cells.     

Increased aerobic metabolism is essential for the beneficial effects of caloric restriction on yeast life span

Calorie restriction is a dietary regimen capable of extending life span in a variety of multicellular organisms. A yeast model of calorie restriction has been developed in which limiting the concentration of glucose in the growth media of Saccharomyces cerevisiae leads to enhanced replicative and chronological longevity. Since S. cerevisiae are Crabtree-positive cells that present repression of aerobic catabolism when grown in high glucose concentrations, we investigated if this phenomenon participates in life span regulation in yeast. S. cerevisiae only exhibited an increase in chronological life span when incubated in limited concentrations of glucose. Limitation of galactose, raffinose or glycerol plus ethanol as substrates did not enhance life span. Furthermore, in Kluyveromyces lactis, a Crabtree-negative yeast, glucose limitation did not promote an enhancement of respiratory capacity nor a decrease in reactive oxygen species formation, as is characteristic of conditions of caloric restriction in S. cerevisiae. In addition, K. lactis did not present an increase in longevity when incubated in lower glucose concentrations. Altogether, our results indicate that release from repression of aerobic catabolism is essential for the beneficial effects of glucose limitation in the yeast calorie restriction model. Potential parallels between these changes in yeast and hormonal regulation of respiratory rates in animals are discussed. G. A. Oliveira and E. B. Tahara contributed equally to this work.     

Aerobic sugar metabolism in the spoilage yeast Zygosaccharomyces bailii

Despite the importance of some Zygosaccharomyces species as agents causing spoilage of food, the carbon and energy metabolism of most of them is yet largely unknown. This is the case with Zygosaccharomyces bailii. In this study the occurrence of the Crabtree effect in the petite-negative yeast Z. bailii ATCC 36947 was investigated. In this yeast the aerobic ethanol production is strictly dependent on the carbon source utilised. In glucose-limited continuous cultures a very low level of ethanol was produced. In fructose-limited continuous cultures ethanol was produced at a higher level and its production increased with the dilution rate. As a consequence, on fructose the onset of respiro-fermentative metabolism caused a reduction in biomass yield. An immediate aerobic alcoholic fermentation in Z. bailii was observed during the transition from sugar limitation to sugar excess, both on glucose and on fructose. The analysis of some key enzymes of the fermentative metabolism showed a high level of acetyl-CoA synthetase in Z. bailii growing on fructose. At high dilution rates, the activities of glucose- and fructose-phosphorylating enzymes, as well as of pyruvate decarboxylase and alcohol dehydrogenase, were higher in cells during growth on fructose than on glucose.     

Tumor cell energy metabolism and its common features with yeast metabolism

During the last decades a considerable amount of research has been focused on cancer. A number of genetic and signaling defects have been identified. This has allowed the design and screening of a number of anti-tumor drugs for therapeutic use. One of the main challenges of anti-cancer therapy is to specifically target these drugs to malignant cells. Recently, tumor cell metabolism has been considered as a possible target for cancer therapy. It is widely accepted that tumors display an enhanced glycolytic activity and oxidative phosphorylation down-regulation (Warburg effect). Therefore, it seems reasonable that disruption of glycolysis might be a promising candidate for specific anti-cancer therapy.     

The function of redox shuttles during aerobic glycolysis in two strains of Ehrlich ascites tumor cells

The function of glycerophosphate and malate-aspartate shuttles during glucose metabolism in two strains of Ehrlich ascites tumor cells was evaluated by several experimental approaches. The activities of the enzymes involved in these shuttle systems were assayed in the cytosolic and mitochondrial compartments after cell fractionation by the digitonin method. The glycerophosphate shuttle can be ruled out because of the lack of relevant enzymatic activities, and the failure of glucose to increase rotenone-inhibited respiration. Analysis of glycolytic flux in the presence of aminooxyacetate indicates that the activity of malate-aspartate shuttle may be very low. Balance studies of glucose uptake and lactate production suggest the existence of other pathways for the reoxidation of cytosolic NADH, which are acetyl-CoA dependent. Estimation of citrate synthase and ATP citrate lyase, in addition to the observed high activity of malate dehydrogenase, suggests a malate-citrate shuttle.     

Oleic acid delays and modulates the transition from respiratory to fermentative metabolism in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> after exposure to glucose excess

This work aimed to study the transition from respiratory to fermentative metabolism in Saccharomyces cerevisiae CEN.PK 113-7D and more specifically to evaluate the implication of the acetyl-coenzymeA-derived carbon transport from cytosol to mitochondria in the onset of the metabolic shift. The strategy consisted in introducing, during aerobic glucose-limited chemostat (D = 0.16 h¹), a local perturbation around the step to be studied by the addition of cosubstrate and in analyzing the consequences of such a perturbation on the metabolic transition. Oleic acid and l-carnitine were among the tested cosubstrates because they were known to stimulate enzymes implicated in the acetyl-coenzymeA transport between the different cell compartments, such as the carnitine acetyl transferases. The metabolic transition was then comparatively quantified in sole glucose and in glucose/oleic acid chemostats in presence/absence of l-carnitine after a pulse of glucose. Feeding the culture with oleic acid (D _ole = 0.0041 and 0.0073 h¹) led to a delay in the onset of the metabolic shift (up to 15 min), a 33% decrease in the ethanol production and a redirection of the carbon flux toward biomass production. The data clearly showed a modulation of the carbon distribution among respiration and fermentation, in favor of a decrease in the “short-term” Crabtree effect by the oleic acid. David Feria-Gervasio and Jean-Roch Mouret worked equally on this project and should be considered both as first authors. An erratum to this article can be found at     

Crabtree/Warburg-like aerobic xylose fermentation by engineered Saccharomyces cerevisiae

Bottlenecks in the efficient conversion of xylose into cost-effective biofuels have limited the widespread use of plant lignocellulose as a renewable feedstock. The yeast Saccharomyces cerevisiae ferments glucose into ethanol with such high metabolic flux that it ferments high concentrations of glucose aerobically, a trait called the Crabtree/Warburg Effect. In contrast to glucose, most engineered S. cerevisiae strains do not ferment xylose at economically viable rates and yields, and they require respiration to achieve sufficient xylose metabolic flux and energy return for growth aerobically. Here, we evolved respiration-deficient S. cerevisiae strains that can grow on and ferment xylose to ethanol aerobically, a trait analogous to the Crabtree/Warburg Effect for glucose. Through genome sequence comparisons and directed engineering, we determined that duplications of genes encoding engineered xylose metabolism enzymes, as well as TKL1, a gene encoding a transketolase in the pentose phosphate pathway, were the causative genetic changes for the evolved phenotype. Reengineered duplications of these enzymes, in combination with deletion mutations in HOG1, ISU1, GRE3, and IRA2, increased the rates of aerobic and anaerobic xylose fermentation. Importantly, we found that these genetic modifications function in another genetic background and increase the rate and yield of xylose-to-ethanol conversion in industrially relevant switchgrass hydrolysate, indicating that these specific genetic modifications may enable the sustainable production of industrial biofuels from yeast. We propose a model for how key regulatory mutations prime yeast for aerobic xylose fermentation by lowering the threshold for overflow metabolism, allowing mutations to increase xylose flux and to redirect it into fermentation products.     

Regulation of protein kinases activity by quercetin in Ehrlich ascites tumor cells

Cyclic AMP-independent protein kinase activities from Ehrlich ascites tumor cells, partially purified by DEAE-cellulose and phosphocellulose chromatography were inhibited by quercetin. The cyclic AMP in the tumor ascites cells and the cyclic AMP-dependent protein kinase activity from this tumor and from bovine and mouse tissues were unaffected by this drug. Since we reported that quercetin elevates cyclic AMP level in Ehrlich ascites tumor cells, this bioflavonoid may have a dual effect on the protein kinae activities in these cells, thus, increasing the cyclic AMP-dependent and decreasing the cyclic AMP-independent protein kinase activities.     

小鼠腹水型肝癌H22a细胞浆内磷蛋白磷酸酶活力调节的研究

小鼠腹水型肝癌细胞胞浆内磷蛋白磷酸酶对磷酸化的组蛋白、酪蛋白、鱼精蛋白具有脱磷酸化活力,而对小分子底物P-Ser、P-Thr、P-Tyr、PNPP等无活力。二价金属离子Mn~(2+)、Co~(2+)、Mg~(2+)对酶有明显激活作用,而Zn~(2+)、F~-、Pi对酶有明显抑制作用。代谢中间物G-6-P、G-1-P、F-6-P、F-1.6-2P、ATP、ADP、GTP对酶有抑制作用,而磷酸化氨基酸和环核苷酸对酶活影响很小。还试验了碱性蛋白质和酸性蛋白质对酶活力的影响,肝素和组蛋白均对酶活力有抑制作用,当两者混和后,其抑制作用会相互抵消。     

Effects of a hexokinase II deletion on the dynamics of glycolysis in continuous cultures of Saccharomyces cerevisiae

In glucose-limited aerobic chemostat cultures of a wild-type Saccharomyces cerevisiae and a derived hxk2 null strain, metabolic fluxes were identical. However, the concentrations of intracellular metabolites, especially fructose 1,6-bisphosphate, and hexose-phosphorylating activities differed. Interestingly, the hxk2 null strain showed a higher maximal growth rate and higher Crabtree threshold dilution rate, revealing a higher oxidative capacity for this strain. After a pulse of glucose, aerobic glucose-limited cultures of wild-type S. cerevisiae displayed an overshoot in the intracellular concentrations of glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-bisphosphate before a new steady state was established, in contrast to the hxk2 null strain which reached a new steady state without overshoot of these metabolites. At low dilution rates the overshoot of intracellular metabolites in the wild-type strain coincided with the immediate production of ethanol after the glucose pulse. In contrast, in the hxk2 null strain the production of ethanol started gradually. However, in spite of the initial differences in ethanol production and dynamic behaviour of the intracellular metabolites, the steady-state fluxes after transition from glucose limitation to glucose excess were not significantly different in the wild-type strain and the hxk2 null strain at any dilution rate.