Effect of growth rate on the glucose metabolism of yeast grown in continuous culture. Radiorespirometric studies

Specifically labelled¹⁴C-d-glucose was used to estimate the percentage participation of glycolysis and pentose phosphate cycle in the glucose catabolism ofCandida utilis andSaccharomyces cerevisiae. The two yeasts were cultivated at various growth rates (0.1 to 0.5 h^?1) in a chemostat on synthetic medium limited with glucose under aerobic conditions. The results show a considerable increase in the percentage participation of pentose phosphate cycle in the glucose catabolized bySaccharomyces cerevisiae with the increase in specific growth rate. However, inCandida utilis, the specific growth rate does not influence significantly the part of glucose catabolized via pentose phosphate cycle, but its absolute values are relatively higher than inSaccharomyces cerevisiae. A rough quantitative estimate indicates that a maximum of 60 to 72% of the assimilated glucose is catabolized through the pentose phosphate cycle while inSaccharomyces cerevisiae the percentage participation of the pentose phosphate cycle varies from 24 to 60% (maximum) and 9 to 34% (minimum).     

Chemostat culture of Bacillus caldotenax at 65°C: Activity and regulation of the main metabolic pathways

Bacillus caldotenax was cultivated in chemostat experiments at 65°C with a chemically defined minimal medium. Glycolysis, tricarboxylic acid cycle, pentose phosphate pathway and the respiratory chain were active as demonstrated by measuring the corresponding enzymes. No enzyme activity of the Entner-Doudoroff pathway could be detected. The specific activities of the citrate cycle enzymes were up to 10 times higher as compared to the enzymes of glycolysis. At dilution rates between 0.3 and 2.2 h^-1 none of the main metabolic pathways was regulated. In contrast the isocitrate lyase was regulated (drop of activity with increasing growth rates). As a result of a batch culture with glucose and acetate as carbon sources a regulation model was proposed: glucose, or a metabolite of glucose, represses the isocitrate lyase; in the absence of glucose acetate acts as an inducer.Abbreviations DCIP dichlorphenol indophenol - ED Entner-Doudoroff pathway - EMP Emden-Meyerhof-Parnas pathway - ICL isocitrate lyase - PP pentose phosphate pathway - TCC tricarbonic acid cycle     

The catabolism of glucose in Tenebrio molitor: The effects of corpora cardiaca

During the development of the mealworm Tenebrio molitor, the effects of corpora cardiaca (CC) extracts on glucose catabolism were tested. In control insects and in insects receiving CC extracts, the activity of the pentose cycle and the glycolytic-citric acid cycle, were evaluated in vivo by a radiorespirometric method using [1-¹⁴C] glucose and [6-¹⁴C] glucose as substrates. The CC extracts strongly divert glucose from the pentose phosphate pathway, which is very active in Tenebrio molitor. Glucose oxidation is reduced by the CC extracts in pupae and adults but is increased in last instar larvae. It seems that the effects of CC extracts vary depending upon the state of carbohydrate reserves.     

Importance of the pentose phosphate pathway for d-glucose catabolism in the obligatory aerobic yeast Rhodotorula gracilis

d-Glucose catabolism of a phosphofructokinase-deficient yeast Rhodotorula gracilis has been studied. By using d-glucose specifically ¹⁴C-labelled at different positions and measuring the distribution of the label in various fractions of cell metabolism, the following results were found. 1. The pentose phosphate pathway, being the main pathway of d-glucose catabolism, simultaneously converts glucose molecules into pentose phosphates oxidatively by using two NADP-linked dehydrogenases and via the non-oxidative transketolase–transaldolase pathway. 2. From the correlation of the ¹⁴CO₂ liberation and the d-glucose consumption and from the fact that the pentose phosphate moiety in nucleic acids is almost equally labelled from d-[1-¹⁴C]- and d-[6-¹⁴C]-glucose, it is concluded that of the glucose utilized about 80% undergoes transformation via the non-oxidative pentose phosphate pathway. Only about 20% of glucose is directly decarboxylated to pentose phosphate. 3. For further degradation it is postulated that the pentose phosphates are split into C₂ fragments and glyceraldehyde 3-phosphates. 4. All three loci of oxidative decarboxylation appear to be effective in Rh. gracilis, the oxidative part of the pentose phosphate pathway, the decarboxylation of pyruvate in the later part of the glycolytic pathway as well as the oxidation in the tricarboxylic acid cycle. 5. d-Glucose molecules taken up are only partially oxidized to CO₂: about four-fifths of each glucose molecule metabolized is incorporated into cell constituents. 6. The quantitative interrelations of the fluxes of d-glucose subunits along the catabolic pathways have been estimated and are discussed.     

The role of glutathione in rat adipocyte pentose phosphate cycle activity

An assay for reduced and oxidized glutathione was adapted to isolated rat epididymal adipocytes in order to correlate pentose phosphate cycle activity and glutathione metabolism. In collagenase-digested adipocytes the [GSH/GSSG] molar ratio was in excess of 100. Cells incubated for 1 hr with low glucose concentrations (0.28–0.55 mm) had higher GSH contents (3.2 μg/10⁶ cells) than in the absence of glucose (2.3 μg/10⁶ cells). The glutathione oxidant diamide caused a dose-related decrease in intracellular GSH, an increase in GSSG released into the medium, but no detectable change in the low intracellular GSSG content. The intracellular content of GSH and amount of GSSG released into the medium were therefore taken to reflect the glutathione status of the adipocytes most closely. Addition of H₂O₂ to a concentration of 60 μm to adipocytes caused to decline within 5 min in GSH content, which was less severe and more rapid to recover in the presence of 1.1 mm glucose, suggesting that the concomitant stimulation of glucose C-1 oxidation induced by the peroxide in the presence of glucose provided NADPH for regeneration of GSH. Further evidence for tight coupling between adipocyte [GSH/GSSG] ratios and pentose phosphate cycle activity was that (i) lowering intracellular GSH to 35–60% of control values by agents as diverse in action as t-butyl hydroperoxide, diamide, or the sulfhydryl blocker N-ethylmaleimide resulted in optimal stimulation of glucose C-1 oxidation and fractional pentose phosphate cycle activity, and (ii) incubating adipocytes directly with 2.5 mm GSSG resulted in a slight increase in glucose C-1 oxidation and when 0.5 mm NADP⁺ was also added a synergistic effect on pentose phosphate cycle activity was found. On the other hand, electron acceptors such as methylene blue did not lower cellular GSH content, but did stimulate the pentose phosphate cycle, confirming a site of action independent of glutathione metabolism. The results show that (i) glucose metabolism by the pentose phosphate cycle contributes to regeneration of GSH and that (ii) glutathione metabolism either directly or via coupled changes in [NADPH/NADP⁺] ratios may play a significant role in short-term control of the pentose phosphate cycle.     

Quantitative determination of the main glucose metabolic fluxes in human erythrocytes by 13C- and 1H-MR spectroscopy.

Information displayed by homonuclear and heteronuclear spin-coupling patterns in 13C- and 1H-MR spectra allowed us to identify the major lactate isotopomers produced either from [1-(13)C]-glucose or from [2-(13)C]-glucose by human erythrocytes. Relative concentrations of detectable isotopomers were determined by integrating the corresponding MR signals. The interpretation of these data in terms of the fractional glucose metabolised through glycolysis and pentose phosphate pathway was performed by a computer simulation of the metabolism that took into account metabolic schemes pertaining to glycolysis and to the F-type of pentose phosphate pathway. The simulation was organised in a way to anticipate the populations of the isotopomers produced from any precursor at a priori established metabolic steady state. By the simulation, isotopomer populations were determined according to different values of pentose cycle, defined as the flux of glyceraldehyde 3-phosphate originating from pentose phosphate pathway at unitary glucose uptake. The populations of the isotopomers originating from [2-(13)C]-glucose were described by polynomials, and ratios between the polynomials were used in conjunction with 13C- and 1H-MR data to determine pentose cycle values. The knowledge of glucose uptake and of pentose cycle value allowed us to perform accurate measurement of the pentose phosphate pathway flux, of the hexokinase and phosphofructokinase fluxes as well as, indirectly, of the carbon dioxide production.     

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.     

Overcoming the metabolic burden of protein secretion in Schizosaccharomyces pombe – A quantitative approach using 13C-based metabolic flux analysis

Protein secretion in yeast is generally associated with a burden to cellular metabolism. To investigate this metabolic burden in Schizosaccharomyces pombe, we constructed a set of strains secreting the model protein maltase in different amounts. We quantified the influence of protein secretion on the metabolism applying ¹³C-based metabolic flux analysis in chemostat cultures. Analysis of the macromolecular biomass composition revealed an increase in cellular lipid content at elevated levels of protein secretion and we observed altered metabolic fluxes in the pentose phosphate pathway, the TCA cycle, and around the pyruvate node including mitochondrial NADPH supply. Supplementing acetate to glucose or glycerol minimal media was found to improve protein secretion, accompanied by an increased cellular lipid content and carbon flux through the TCA cycle as well as increased mitochondrial NADPH production. Thus, systematic metabolic analyses can assist in identifying factors limiting protein secretion and in deriving strategies to overcome these limitations.     

Reduced Oxidative Pentose Phosphate Pathway Flux in Recombinant Xylose-Utilizing Saccharomyces cerevisiae Strains Improves the Ethanol Yield from Xylose

In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD⁺. In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g⁻¹) and the lowest xylitol yield (0.05 g g⁻¹) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.     

Metabolic network reconstruction,growth characterization and 13C-metabolic flux analysis of the extremophile Thermus thermophilus HB8

Thermus thermophilus is an extremely thermophilic bacterium with significant biotechnological potential. In this work, we have characterized aerobic growth characteristics of T. thermophilus HB8 at temperatures between 50 and 85 °C, constructed a metabolic network model of its central carbon metabolism and validated the model using ¹³C-metabolic flux analysis (¹³C–MFA). First, cells were grown in batch cultures in custom constructed mini-bioreactors at different temperatures to determine optimal growth conditions. The optimal temperature for T. thermophilus grown on defined medium with glucose was 81 °C. The maximum growth rate was 0.25 h⁻¹. Between 50 and 81 °C the growth rate increased by 7-fold and the temperature dependence was described well by an Arrhenius model with an activation energy of 47 kJ/mol. Next, we performed a ¹³C-labeling experiment with [1,2-¹³C] glucose as the tracer and calculated intracellular metabolic fluxes using ¹³C–MFA. The results provided support for the constructed network model and highlighted several interesting characteristics of T. thermophilus metabolism. We found that T. thermophilus largely uses glycolysis and TCA cycle to produce biosynthetic precursors, ATP and reducing equivalents needed for cells growth. Consistent with its proposed metabolic network model, we did not detect any oxidative pentose phosphate pathway flux or Entner-Doudoroff pathway activity. The biomass precursors erythrose-4-phosphate and ribose-5-phosphate were produced via the non-oxidative pentose phosphate pathway, and largely via transketolase, with little contribution from transaldolase. The high biomass yield on glucose that was measured experimentally was also confirmed independently by ¹³C–MFA. The results presented here provide a solid foundation for future studies of T. thermophilus and its metabolic engineering applications.     

Metabolic Control of Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)-mediated Caspase-2 Suppression by the B55β/Protein Phosphatase 2A (PP2A)

High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway. This metabolic control is exerted via inhibitory phosphorylation of the caspase-2 prodomain by activated Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). We show here that this activation of CaMKII depends, in part, on dephosphorylation of CaMKII at novel sites (Thr³⁹³/Ser³⁹⁵) and that this is mediated by metabolic activation of protein phosphatase 2A in complex with the B55β targeting subunit. This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and protein phosphatase 2A-regulated physiological processes, because both enzymes appear to be responsive to alterations in glucose metabolized via the pentose phosphate pathway.     

Hierarchy in Pentose Sugar Metabolism in Clostridium acetobutylicum

Bacterial metabolism of polysaccharides from plant detritus into acids and solvents is an essential component of the terrestrial carbon cycle. Understanding the underlying metabolic pathways can also contribute to improved production of biofuels. Using a metabolomics approach involving liquid chromatography-mass spectrometry, we investigated the metabolism of mixtures of the cellulosic hexose sugar (glucose) and hemicellulosic pentose sugars (xylose and arabinose) in the anaerobic soil bacterium Clostridium acetobutylicum. Simultaneous feeding of stable isotope-labeled glucose and unlabeled xylose or arabinose revealed that, as expected, glucose was preferentially used as the carbon source. Assimilated pentose sugars accumulated in pentose phosphate pathway (PPP) intermediates with minimal flux into glycolysis. Simultaneous feeding of xylose and arabinose revealed an unexpected hierarchy among the pentose sugars, with arabinose utilized preferentially over xylose. The phosphoketolase pathway (PKP) provides an alternative route of pentose catabolism in C. acetobutylicum that directly converts xylulose-5-phosphate into acetyl-phosphate and glyceraldehyde-3-phosphate, bypassing most of the PPP. When feeding the mixture of pentose sugars, the labeling patterns of lower glycolytic intermediates indicated more flux through the PKP than through the PPP and upper glycolysis, and this was confirmed by quantitative flux modeling. Consistent with direct acetyl-phosphate production from the PKP, growth on the pentose mixture resulted in enhanced acetate excretion. Taken collectively, these findings reveal two hierarchies in clostridial pentose metabolism: xylose is subordinate to arabinose, and the PPP is used less than the PKP.     

Carbon-Flux Distribution within Streptomyces coelicolor Metabolism: A Comparison between the Actinorhodin-Producing Strain M145 and Its Non-Producing Derivative M1146

Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two Streptomyces coelicolor A3 (2) strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and ¹³C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest.     

Fermentation of Xylose Causes Inefficient Metabolic State Due to Carbon/Energy Starvation and Reduced Glycolytic Flux in Recombinant Industrial Saccharomyces cerevisiae

In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3- phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7- phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4. These results provide a metabolic explanation for the low ethanol productivity on xylose compared to glucose.     

Transfer of 1-pyrroline-5-carboxylate as oxidizing potential from hepatocytes to erythrocytes.

The interconversions of proline and 1-pyrroline-5-carboxylate form an intercellular cycle that is the basis of a metabolic interaction between hepatocytes and erythrocytes. The cycle transfers oxidizing potential from hepatocytes to erythrocytes, which stimulates pentose phosphate pathway in erythrocytes. This interaction depends on the differential metabolism of proline and 1-pyrroline-5-carboxylate in erythrocytes and hepatocytes and consists of the following: in hepatocytes proline oxidase converts proline into 1-pyrroline-5-carboxylate, which is released into the medium and taken up by erythrocytes; erythrocyte 1-pyrroline-5-carboxylate reductase converts 1-pyrroline-5-carboxylate into proline and concomitantly generates NADP+; the generated oxidizing potential drives glucose metabolism through the pentose phosphate pathway in erythrocytes; finally, erythrocytes release proline into the medium, enabling it to re-enter hepatocytes and repeat the cycle. The increased activity of the pentose phosphate pathway in erythrocytes may enhance the production of 5-phosphoribosyl pyrophosphate, a necessary moiety for the processing of purines.     

Effect of Water Stress on the Carbohydrate Metabolism of Citrullus lanatus Seeds during Germination

Gluconeogenesis in Citrullus lanatus seeds is a post germinative event. Increases in isocitrate lyase activity and incorporation of radioactivity from [2-¹⁴C]acetate into sugars occur only after radicle emergence. During germination, the seeds appear to rely on carbohydrate as the respiratory substrate. At this time, glycolysis, the pentose phosphate pathway, and the tricarbocyclic acid cycle seem to be functional. Utilization of raffinose during germination appears to be important.

Water stress, which completely inhibits germination, has a marked effect on carbohydrate metabolism. The rate of ¹⁴CO₂ release from [2-¹⁴C]acetate, [1-¹⁴C]glucose, and [6-¹⁴C]glucose is lower in the stressed seeds than the control seeds during the respiratory lag phase. However, in the stressed seeds neither glycolysis, the pentose phosphate pathway, nor the tricarboxylic acid cycle is completely inhibited. In contrast to the control seeds in which raffinose content sharply declines after 12 h of incubation, raffinose content in the stressed seeds remains fairly constant.

The respiratory lag phase of the control seeds coincides with a lower reducing substance content, glucose content, and fructose content than in the stressed seeds during the corresponding incubation period.

     

Sucrose and starch metabolism in carrot (Daucus carota L.) cell suspensions analysed by 13-labelling: indications for a cytosol and a plastid-localized oxidative pentose phosphate pathway

Cells were grown in batch culture on a mixture of 50 mM glucose and fructose as the carbon source; either the glucose or the fructose was [1-¹³C]-labelled. In order to investigate the uptake and conversion of glucose and fructose during long-term labelling experiments in cell suspensions of Daucus carota L., samples were taken every 2 d during a 2 week culture period and sucrose and starch were assayed by means of HPLC and ¹³C-nuclear magnetic resonance. The fructose moieties of sucrose had a lower labelling percentage than the glucose moieties. Oxidative pentose phosphate pathway activity in the cytosol is suggested to be responsible for this loss of label of especially C-1 carbons. A combination of oxidative pentose phosphate pathway activity, a relatively high activity of pathway to sucrose synthesis and a slow equilibration between glucose-6-phosphate and fructose-6-phosphate could explain these results. Starch contained glucose units with a much lower labelling percentage than glucose moieties of sucrose: it was concluded that a second, plastid-localized, oxidative pentose phosphate pathway was responsible for removal of C-1 carbons of the glucosyl units used for synthesis of starch. Redistribution of label from [1-¹³C]-hexoses to [6-¹³C]-hexoses also occurred: 18-45% of the label was found at the C-6 carbons. This is a consequence of cycling between hexose phosphates and those phosphates in the cytosol catalysed by PFP. The results indicate that independent (oxidative pentose phosphate pathway mediated) sugar converting cycles exist in the cytosol and plastid.Key words: Daucus carotaL., cell suspensions, carbon-13 nuclear magnetic resonance, ¹³C-NMR, carbohydrate cycling, oxidative pentose phosphate pathway, plastid.      

Metabolism of glucose and gluconate in fast- and slow-growing rhizobia

Enzymatic evidence was sought for the operation of pathways involved in glucose and gluconate catabolisms in fast- and slow-growing Rhizobium species including members of the cowpea group. Enzymes of the Entner-Doudoroff pathway, pentose phosphate pathway and tricarboxylic acid cycle were detected in fast-growing rhizobia but the pentose phosphate pathway was absent in slow-growers, regardless of the carbon source used. When analysed for enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways in glucose-grown cells, the pathways were found to operate simultaneously in rhizobia.     

Interrelationship and control of glucose metabolism and lipogenesis in isolated fat-cells. Control of pentose phosphate-cycle activity by cellular requirement for reduced nicotinamide adenine dinucleotide phosphate

By using inhibitors and stimulators of different metabolic pathways the interdependence of the pentose phosphate cycle and lipogenesis in isolated fat-cells was studied. Rotenone, which is known to inhibit electron transport in the respiratory chain, blocked glucose breakdown at the site of pyruvate dehydrogenase. Consequently, because of the lack of acetyl-CoA, fatty acid synthesis was almost abolished. A concomitant decrease in pentose phosphate-cycle activity was observed. Phenazine methosulphate stimulated pentose phosphate-cycle activity about five- to ten-fold without a considerable effect on fatty acid synthesis. The influence of rotenone on both the pentose phosphate cycle and lipogenesis could be overcome by addition of phenazine methosulphate, indicating that rotenone has no direct effect on these pathways. The decreased rate of the pentose phosphate cycle in the presence of rotenone therefore has to be considered as a consequence of decreased fatty acid synthesis. The rate of glucose catabolism via the pentose phosphate cycle in adipocytes appears to be determined by the requirement of NADPH for lipogenesis. Treatment of cells with 6-aminonicotinamide caused an accumulation of 6-phosphogluconate, indicating an inhibition of 6-phosphogluconate dehydrogenase. The rate of glucose metabolism via the pentose phosphate cycle as well as the rate of fatty acid synthesis, however, was not affected by 6-aminonicotinamide treatment and could still be stimulated by addition of insulin. Since even in cells from starved animals, in which the pentose phosphate-cycle activity is extremely low, no accumulation of 6-phosphogluconate was observed, it is concluded that the control of this pathway is achieved by the rate of regeneration of NADP at the site of glucose 6-phosphate dehydrogenase.     

Re-examination of metabolic fluxes in <Emphasis Type="Italic">Escherichia coli</Emphasis> during anaerobic fermentation of glucose using <Superscript>13</Superscript>C labeling experiments and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy

Improved design of metabolic flux estimation using mixed label ¹³C labeling experiments and identifiability analysis motivated re-examination of metabolic fluxes during anaerobic fermentation in the Escherichia coli. Comprehensive metabolic flux maps were determined by using a mixture of differently labeled glucose and compared to conventional flux maps obtained using extracellular measurements and comprehensive metabolic flux maps obtained using only U-¹³C glucose as the substrate. As expected, conventional flux analysis performs poorly in comparison to ¹³C-MFA, especially in the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways. Identifiability analysis indicated and experiments confirmed that a mixture of 10% U-^l3C glucose, 25% 1-¹³C glucose, and 65% naturally labeled glucose significantly improved the statistical quality of all calculated fluxes in the PP pathway, the EMP pathway, the anaplerotic reactions, and the tricarboxylic acid cycle. Modifying the network topology for the presence and absence of the Entner-Doudoroff pathway and the glyoxylate shunt did not affect the value or quality of estimated fluxes significantly. Extracellular measurement of formate production was necessary for the accurate estimation of the fluxes around the formate node.