Concentrations Of Glycolytic Intermediates Research Articles

Metabolic Dysregulation and Neurovascular Dysfunction in Diabetic Retinopathy.

Diabetic retinopathy is a major cause of ocular complications in patients with type 1 and type 2 diabetes in developed countries. Due to the continued increase in the number of people with obesity and diabetes in the United States of America and globally, the incidence of diabetic retinopathy is expected to increase significantly in the coming years. Diabetic retinopathy is widely accepted as a combination of neurodegenerative and microvascular changes; however, which change occurs first is not yet understood. Although the pathogenesis of diabetic retinopathy is very complex, regulated by numerous signaling pathways and cellular processes, maintaining glucose homeostasis is still an essential component for normal physiological functioning of retinal cells. The maintenance of glucose homeostasis is finely regulated by coordinated interplay between glycolysis, Krebs cycle, and oxidative phosphorylation. Glycolysis is the most conserved metabolic pathway in biology and is tightly regulated to maintain a steady-state concentration of glycolytic intermediates; this regulation is called scheduled or regulated glycolysis. However, an abnormal increase in glycolytic flux generates large amounts of intermediate metabolites that can be shunted into different damaging pathways including the polyol pathway, hexosamine pathway, diacylglycerol-dependent activation of the protein kinase C pathway, and Amadori/advanced glycation end products (AGEs) pathway. In addition, disrupting the balance between glycolysis and oxidative phosphorylation leads to other biochemical and molecular changes observed in diabetic retinopathy including endoplasmic reticulum-mitochondria miscommunication and mitophagy dysregulation. This review will focus on how dysregulation of glycolysis contributes to diabetic retinopathy.

The dynamic side of the Warburg effect: glycolytic intermediate storage as buffer for fluctuating glucose and O 2supply in tumor cells.

Background: Tumor cells often show altered metabolism which supports uncontrolled proliferation. A classic example is the Warburg effect: high glucose uptake and lactate production despite sufficient oxygen supply. Remarkably, tumor cells can transiently take up glucose even an order of magnitude faster when glucose is reintroduced after depletion. Regulation and significance of this high glucose uptake are investigated here. Methods: A new computational model was developed which reproduces two types of experimental data on Ehrlich ascites tumor cells: measurements by Otto Warburg of the average aerobic glycolytic rate during one hour (Warburg effect), and fast metabolic responses measured by others during the first minutes after reintroducing glucose. The model is subsequently extended with equations for glucose and O 2transport to predict the role of metabolism during fluctuations of blood flow in tumor tissue. Results: Model analysis reveals dynamic regulation of the head section of glycolysis where glucose uptake and phosphorylation occur. The head section is disinhibited slowly when concentrations of glycolytic intermediates fall, causing glucose uptake rate to considerably exceed that found by Warburg. The head section is partially inhibited in about a minute when sufficient glucose has been taken up. Simulations predict that tumors greedily take up glucose when blood flow resumes after periods of low flow. The cells then store glucose as fructose 1,6-bisphosphate and other glycolytic intermediates. During subsequent periods of low flow that cause O 2 and glucose depletion these stores are used for ATP production and biomass. Conclusions: The powerful glycolytic system in tumors not only synthesizes ATP at high steady rates, but can also store glycolytic intermediates to buffer temporary oxygen and nutrient shortages for up to 10 minutes. The head section of glycolysis in tumor cells, disinhibited during glucose shortages, becomes very efficient at stealing glucose from other cells, even at low glucose concentrations.

The dynamic side of the Warburg effect: glycolytic intermediate storage as buffer for fluctuating glucose and O 2 supply in tumor cells

Background: Tumor cells often show altered metabolism which supports uncontrolled proliferation. A classic example is the Warburg effect: high glucose uptake and lactate production despite sufficient oxygen supply. Remarkably, tumor cells can transiently take up glucose even an order of magnitude faster when glucose is reintroduced after depletion. Regulation and significance of this high glucose uptake are investigated here. Methods: A new computational model was developed which reproduces two types of experimental data on Ehrlich ascites tumor cells: measurements by Otto Warburg of the average aerobic glycolytic rate during one hour (Warburg effect), and fast metabolic responses measured by others during the first minutes after reintroducing glucose. The model is subsequently extended with equations for glucose and O 2 transport to predict the role of metabolism during fluctuations of blood flow in tumor tissue. Results: Model analysis reveals dynamic regulation of the head section of glycolysis where glucose uptake and phosphorylation occur. The head section is disinhibited slowly when concentrations of glycolytic intermediates fall, causing glucose uptake rate to considerably exceed that found by Warburg. The head section is partially inhibited in about a minute when sufficient glucose has been taken up. Simulations predict that tumors greedily take up glucose when blood flow resumes after periods of low flow. The cells then store glucose as fructose 1,6-bisphosphate and other glycolytic intermediates. During subsequent periods of low flow that cause O 2 and glucose depletion these stores are used for ATP production and biomass. Conclusions: The powerful glycolytic system in tumors not only synthesizes ATP at high steady rates, but can also store glycolytic intermediates to buffer temporary oxygen and nutrient shortages for up to 10 minutes. The head section of glycolysis in tumor cells, disinhibited during glucose shortages, becomes very efficient at stealing glucose from other cells, even at low glucose concentrations.

The dynamic side of the Warburg effect: glycolytic intermediates as buffer for fluctuating glucose and O2 supply in tumor cells

Background:Tumor cells show the Warburg effect: high glucose uptake and lactate production despite sufficient oxygen supply. Otto Warburg found this effect in tissue slices and in suspensions of Ehrlich ascites tumor cells. Remarkably, these ascites tumor cells can transiently take up glucose an order of magnitude faster than the steady high rate measured by Warburg for hours.Methods:The purpose of the transiently very high glucose uptake is investigated here with a computational model of glycolysis, oxidative phosphorylation and ATP consumption which reproduces short kinetic experiments on the ascites tumor cells as well as the long-lasting Warburg, Crabtree and Pasteur effects. The model, extended with equations for glucose and O2 transport in tissue, is subsequently used to predict metabolism in tumor cells during fluctuations of tissue blood flow resulting in cycling hypoxia.Results:The model analysis suggests that the head section of the glycolytic chain in the tumor cells is partially inhibited in about a minute when substantial amounts of glucose have been taken up intracellularly; this head section of the glycolytic chain is subsequently disinhibited slowly when concentrations of glycolytic intermediates are low. Based on these dynamic characteristics, simulations of tissue with fluctuating O2and glucose supply predict that tumor cells greedily take up glucose when this periodically becomes available, leaving very little for other cells. The glucose is stored as fructose 1,6-bisphosphate and other glycolytic intermediates, which are used for ATP production during O2and glucose shortages.Conclusions:The head section of glycolysis which phosphorylates glucose may be dynamically regulated and takes up glucose at rates exceeding the Warburg effect if glucose levels have been low for some time. The hypothesis is put forward here that dynamic regulation of the powerful glycolytic enzyme system in tumors is used to buffer oxygen and nutrient fluctuations in tissue.

CELL BIOLOGY SYMPOSIUM: Practical application of the basic aspects of GLUT4 membrane trafficking and insulin signaling on issues related to animal agriculture.

Because of the relatively short lifespans of beef cattle, membrane trafficking in relation to inflammation is not considered important unless it overtly affects productivity. However, glucose uptake and utilization is important for adipose tissue development in beef cattle, and increasing glucose utilization in intramuscular adipose tissue can increase carcass quality. Research from the 1980s demonstrated a lack of insulin sensitivity in isolated bovine adipocytes and adipose tissue explants incubated in vitro. Insulin did not stimulate glucose or acetate incorporation into fatty acids, nor did it increase concentrations of glycolytic intermediates in bovine adipose tissue incubated with exogenous glucose. Specific binding of [I] iodoinsulin and insulin degradation in bovine isolated adipocytes was low to non-detectable. These early studies indicated that insulin-dependent receptor-mediated signaling was less important in bovine adipose tissue than in adipose tissues of humans, swine, or laboratory species. More recent research demonstrated that glucose transporter protein 4 (GLUT4) expression in muscle and adipose tissue declines markedly after birth in calves, indicating the development of insulin resistance as cattle transition from suckling to functional ruminants. Insulin resistance is important in dairy cattle, causing ketosis and fatty liver. Consistent with this, subcutaneous adipose tissue expression decreases 50% following parturition in dairy cattle, although expression of genes associated with insulin responsiveness (, , and ) is up-regulated by 21 d postpartum. Understanding the underlying mechanisms of insulin resistance in beef and dairy cattle would increase animal health and thereby improve productivity.

S100B impairs glycolysis via enhanced poly(ADP-ribosyl)ation of glyceraldehyde-3-phosphate dehydrogenase in rodent muscle cells

S100 calcium-binding protein B (S100B), a multifunctional macromolecule mainly expressed in nerve tissues and adipocytes, has been suggested to contribute to the pathogenesis of obesity. To clarify the role of S100B in insulin action and glucose metabolism in peripheral tissues, we investigated the effect of S100B on glycolysis in myoblast and myotube cells. Rat myoblast L6 cells were treated with recombinant mouse S100B to examine glucose consumption, lactate production, glycogen accumulation, glycolytic metabolites and enzyme activity, insulin signaling, and poly(ADP-ribosyl)ation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Glycolytic metabolites were investigated by enzyme assays or metabolome analysis, and insulin signaling was assessed by Western blot analysis. Enzyme activity and poly(ADP-ribosyl)ation of GAPDH was evaluated by an enzyme assay and immunoprecipitation followed by dot blot with an anti-poly(ADP-ribose) antibody, respectively. S100B significantly decreased glucose consumption, glucose analog uptake, and lactate production in L6 cells, in either the presence or absence of insulin. In contrast, S100B had no effect on glycogen accumulation and insulin signaling. Metabolome analysis revealed that S100B increased the concentration of glycolytic intermediates upstream of GAPDH. S100B impaired GAPDH activity and increased poly(ADP-ribosyl)ated GAPDH proteins. The effects of S100B on glucose metabolism were mostly canceled by a poly(ADP-ribose) polymerase inhibitor. Similar results were obtained in C2C12 myotube cells. We conclude that S100B as a humoral factor may impair glycolysis in muscle cells independent of insulin action, and the effect may be attributed to the inhibition of GAPDH activity from enhanced poly(ADP-ribosyl)ation of the enzyme.

Altered Kinetics Properties of Erythrocyte Lactate Dehydrogenase in Type II Diabetic Patients and Its Implications for Lactic Acidosis.

Recent studies have been noted that the erythrocytes from Type II diabetic patients show significantly altered structural and functional characteristics along with the changed intracellular concentrations of glycolytic intermediates. More recent studies from our laboratory have shown that the activities of enzymes of glycolytic pathway changed significantly in RBCs from Type II diabetic patients. In particular the levels of lactate dehydrogenase (LDH) increased significantly. Lactic acidosis is an established feature of diabetes and LDH plays a crucial role in conversion of pyruvate to lactate and reportedly, the levels of lactate are significantly high which is consistent with our observation on increased levels of LDH. Owing to this background, we examined the role of erythrocyte LDH in lactic acidosis by studying its kinetics properties in Type II diabetic patients. Km, Vmax and apparent catalytic efficiency were determined using pyruvate and NADH as the substrates. With pyruvate as the substrate the Km values were comparable but Vmax increased significantly in the diabetic group. With NADH as the substrate the enzyme activity of the diabetic group resolved in two components as against a single component in the controls. The Apparent Kcat and Kcat/Km values for pyruvate increased in the diabetic group. The Ki for pyruvate increased by two fold for the enzyme from diabetic group with a marginal decrease in Ki for NADH. The observed changes in catalytic attributes are conducive to enable the enzyme to carry the reaction in forward direction towards conversion of pyruvate to lactate leading to lactic acidosis.

α-ketoglutarate coordinates carbon and nitrogen utilization via enzyme I inhibition

Microbes survive in a variety of nutrient environments by modulating their intracellular metabolism. Balanced growth requires coordinated uptake of carbon and nitrogen, the primary substrates for biomass production. The mechanisms that balance carbon and nitrogen uptake are, however, poorly understood. We find in Escherichia coli that a sudden increase in nitrogen availability results in an almost immediate increase in glucose uptake. The concentrations of known glycolytic intermediates and regulators, however, remain homeostatic. Instead, we find that α-ketoglutarate, which accumulates in nitrogen limitation, directly blocks glucose uptake by inhibiting Enzyme I, the first step of the phosphotransferase system (PTS). This enables rapid modulation of glycolytic flux without marked concentration changes in glycolytic intermediates by simultaneously accelerating glucose import and consumption of the terminal glycolytic intermediate phosphoenolpyruvate. Quantitative modeling shows that this previously unidentified regulatory connection is in principle sufficient to coordinate carbon and nitrogen utilization.

13C‐metabolic flux analysis for batch culture of Escherichia coli and its pyk and pgi gene knockout mutants based on mass isotopomer distribution of intracellular metabolites

Since most bio-production processes are conducted in a batch or fed-batch manner, the evaluation of metabolism with respect to time is highly desirable. Toward this aim, we applied (13)C-metabolic flux analysis to nonstationary conditions by measuring the mass isotopomer distribution of intracellular metabolites. We performed our analysis on batch cultures of wild-type Escherichia coli, as well as on Pyk and Pgi mutants, obtained the fluxes and metabolite concentrations as a function of time. Our results for the wild-type indicated that the TCA cycle flux tended to increase during growth on glucose. Following glucose exhaustion, cells controlled the branch ratio between the glyoxylate pathway and the TCA cycle, depending on the availability of acetate. In the Pyk mutant, the concentrations of glycolytic intermediates changed drastically over time due to the dumping and feedback inhibition caused by PEP accumulation. Nevertheless, the flux distribution and free amino acid concentrations changed little. The growth rate and the fluxes remained constant in the Pgi mutant and the glucose-6-phosphate dehydrogenase reaction was the rate-limiting step. The measured fluxes were compared with those predicted by flux balance analysis using maximization of biomass yield or ATP production. Our findings indicate that the objective function of biosynthesis became less important as time proceeds on glucose in the wild-type, while it remained highly important in the Pyk mutant. Furthermore, ATP production was the primary objective function in the Pgi mutant. This study demonstrates how cells adjust their metabolism in response to environmental changes and/or genetic perturbations in the batch cultivation.

Changes in the Concentrations of Glycolytic Intermediates and Nucleotides and Their Relation to Phosphofructokinase Activity in the Liver of Yellowtail

Changes in the Concentrations of Glycolytic Intermediates and Nucleotides and Their Relation to Phosphofructokinase Activity in the Liver of Yellowtail

Quantitative Metabolome Profiling of Colon and Stomach Cancer Microenvironment by Capillary Electrophoresis Time-of-Flight Mass Spectrometry

Most cancer cells predominantly produce energy by glycolysis rather than oxidative phosphorylation via the tricarboxylic acid (TCA) cycle, even in the presence of an adequate oxygen supply (Warburg effect). However, little has been reported regarding the direct measurements of global metabolites in clinical tumor tissues. Here, we applied capillary electrophoresis time-of-flight mass spectrometry, which enables comprehensive and quantitative analysis of charged metabolites, to simultaneously measure their levels in tumor and grossly normal tissues obtained from 16 colon and 12 stomach cancer patients. Quantification of 94 metabolites in colon and 95 metabolites in stomach involved in glycolysis, the pentose phosphate pathway, the TCA and urea cycles, and amino acid and nucleotide metabolisms resulted in the identification of several cancer-specific metabolic traits. Extremely low glucose and high lactate and glycolytic intermediate concentrations were found in both colon and stomach tumor tissues, which indicated enhanced glycolysis and thus confirmed the Warburg effect. Significant accumulation of all amino acids except glutamine in the tumors implied autophagic degradation of proteins and active glutamine breakdown for energy production, i.e., glutaminolysis. In addition, significant organ-specific differences were found in the levels of TCA cycle intermediates, which reflected the dependency of each tissue on aerobic respiration according to oxygen availability. The results uncovered unexpectedly poor nutritional conditions in the actual tumor microenvironment and showed that capillary electrophoresis coupled to mass spectrometry-based metabolomics, which is capable of quantifying the levels of energy metabolites in tissues, could be a powerful tool for the development of novel anticancer agents that target cancer-specific metabolism.

Substrate utilization and dose response to insulin by subcutaneous adipose tissue of Angus steers fed corn- or hay-based diets

We hypothesized that, at a common age endpoint, adipose tissue from corn-fed steers would be less sensitive to insulin than adipose tissue from hay-fed steers. Angus steers were assigned to either a corn-based diet (n = 6) or hay-based diet (n = 6) and fed to common days on feed. Steers fed the corn-based diet had 2.44 cm of fat thickness over the 12th thoracic rib, whereas hay-fed steers had 1.04 cm of fat thickness. At slaughter, subcutaneous adipose samples were collected and portions of subcutaneous adipose tissue were incubated with [U-(14)C]acetate to quantify fatty acid synthesis or with [U-(14)C]glucose to assess glucose utilization in the presence of 0, 100, or 500 ng/mL of insulin. Additional subcutaneous samples were used to evaluate glycolytic intermediate concentrations as indicators of glycolytic flux. Data were analyzed as a split-plot with diet in the main plot and insulin concentration and its interaction with diet in the sub-plot. Within diet, linear and quadratic contrasts of insulin concentration were tested. Diet had no effect (P > or = 0.31) on glucose metabolism or acetate carbon incorporation into total lipids (P = 0.32). Insulin had no effect (P > 0.21) on glucose conversion to CO(2), lactate, or total lipids, nor did it affect (P = 0.28) acetate conversion to total lipids. No diet x insulin interaction (P > 0.36) was observed for any measure of subcutaneous metabolism in vitro. Steers fed the corn-based diet exhibited neither a linear (P > 0.22) nor a quadratic (P > 0.24) effect to increasing insulin concentration. However, when steers were fed the hay-based diet, there was a positive linear (P = 0.06) effect for glucose oxidation. These results suggest that subcutaneous adipose tissue may become resistant to stimulation by insulin in steers fed to a fat thickness above the average feedlot steer, but this is independent of diet.

Metabolic profiling of Escherichia coli cultivations: evaluation of extraction and metabolite analysis procedures

The quantitative estimation of intracellular metabolite concentrations (metabolic profiling) is a prerequisite for a better understanding of biological processes and thus inevitable for the rational improvement of microbial production strains and process design. Since pool sizes of substrates regulate flux through different enzymes, the accurate determination of intracellular metabolite concentrations is necessary to understand in vivo reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates in Escherichia coli K12 was achieved by using a novel in situ rapid sampling and quenching procedure. A new extraction procedure using buffered hot water was established. By use of simultaneous multi-substrate feeding with various ratios of glucose, fructose and acetate during continuous cultivations several metabolic states were induced. Metabolic flux analysis and the newly developed metabolic profiling procedure were used to determine in vivo enzyme kinetics as exemplified for fructose 1,6-bisphosphate aldolase and citrate synthase.

The dimorphic yeast Yarrowia lipolytica possesses an atypical phosphofructokinase: characterization of the enzyme and its encoding gene

The phosphofructokinase from the non-conventional yeast Yarrowia lipolytica (YlPfk) was purified to homogeneity, and its encoding gene isolated. YlPfk is an octamer of 869 kDa composed of a single type of subunit, and shows atypical kinetic characteristics. It did not exhibit cooperative kinetics for fructose 6-phosphate (Hill coefficient, h 1.1; S0.5 52 microM), it was inhibited moderately by MgATP (Ki 3.5 mM), and it was strongly inhibited by phosphoenolpyruvate (Ki 61 microM). Fructose 2,6-bisphosphate did not activate the enzyme, and AMP and ADP were also without effect. The gene YlPFK1 has no introns, and encodes a putative protein of 953 aa, with a molecular mass consistent with the subunit size found after purification. Disruption of the gene abolished growth in glucose and Pfk activity, while reintroduction of the gene restored both properties. This indicates that Y. lipolytica has only one gene encoding Pfk, and supports the finding that the enzyme consists of identical subunits. Glucose did not interfere with growth of the Ylpfk1 disruptant in permissive carbon sources. The unusual kinetic characteristics of YlPfk, and the intracellular concentrations of glycolytic intermediates during growth in glucose, suggest that YlPfk may play an important role in the regulation of glucose metabolism in Y. lipolytica, different from the role played by the enzyme in Saccharomyces cerevisiae.

Postmortem regulation of glycolysis by 6-phosphofructokinase in bovine M. Sternocephalicus pars mandibularis

This experiment addressed the hypothesis that 6-phosphofructokinase (6-PFK) regulates glycolysis in postmortem in M. sternocephalicus pars mandibularis. In two separate experiments, muscle samples were excised from randomly-selected steers that would typically be found on a commercial slaughter floor. In the first experiment, two samples were obtained from each of 6 steers immediately post-exsanguination; one sample was immersed immediately in liquid nitrogen and the other was stored at 4 °C for 4 d, to compare 6-PFK enzyme activity and glycolytic intermediate concentrations between fresh and d 4 postmortem samples. The greatest activity of 6-PFK was measured in fresh muscle extracts at pH 7.4, whereas little activity was detectable at pH 7.0. 6-PFK activity measured at pH 7.4 in d 4 samples also was barely detectable. Hill coefficient values for 6-PFK in fresh samples measured at pH 7.4 or 7.0, and d 4 samples measured at pH 7.4 were 2.9, 0.8, and 0.7, respectively, indicating loss of cooperativity with both lowered pH during assay and with time postmortem. Glycogen concentrations decreased 45% from d 0 to d 4, to 39.6 μmol glycogen/g muscle. Muscle concentrations of free glucose increased ( P < 0.001) from 0.84 μmol/g at d 0 to 6.54 μmol/g at d 4. Fructose-6-phosphate and glucose-6-phosphate increased ( P < 0.001) from d 0 to d 4 (2.8-fold and 4.7-fold, respectively). Lactate began accumulating immediately (3.33 μmol/g) and was elevated to 45.9 μmol/g by d 4. In the second experiment, conversion of [U- 14C]glucose to lactate, glycogen, and CO 2 was measured in vitro at pH 7.4 and 7.0 in fresh M. sternocephalicus pars mandibularis strips from four steers. Total [U- 14C]glucose was less in muscle strips incubated at pH 7.0 than in those incubated at pH 7.4 (55.5 vs. 123 nmol glucose utilized per 100 mg muscle per h; P = 0.04), due primarily to a reduction in glucose conversion to lactate. The conversion of glucose to glycogen or CO 2 in vitro was unaffected by media pH. These results suggest that the postmortem decline in pH in M. sternocephalicus pars mandibularis ultimately inactivates 6-PFK; this occurs prior to the depletion of glycogen reserves.

Ex Vivo Treatment of Erythroid Cells with Glycolytic Intermediates for Metabolic Correction of Pyruvate Kinase Deficiency.

Glycolysis is an essential metabolic pathway for survival of red blood cells (RBC), and mutations in the RBC-type of pyruvate kinase (R-PK) cause hereditary non-spherocytic hemolytic anemia. We have shown that R-PK deficiency induced apoptosis in erythroid progenitor cells (Blood 96: 596a, 2000; Am J Hematol 74: 68, 2003). In this study, we examined whether ex vivo treatment of erythroid cells with high concentration of glycolytic intermediates ameliorated apoptosis of R-PK deficiency. For this purpose, we used the R-PK deficient Friend erythroleukemic cell, SLC3, which has been established from a mice model of R-PK deficiency (Jpn J Cancer Res 90: 171, 1999). SLC3 and a control Friend cell were cultured with 0, 1, 3, and 5mM phosphoenolpyruvate (PEP) or pyruvate (PA). Supplementations of either PEP or PA resulted in about 2-fold accumulation of intracellular PA; however, ATP concentration was not significantly changed. Flow cytometric analysis showed that 24-hours treatment with 5mM PEP significantly reduced annexinV (+)/PI (−) cells. These data suggested that exogenously supplemented glycolytic intermediates increased intracellular PA concentration, resulting in amelioration of erythroid apoptosis. We have shown that a glutathione precursor, N-acetyl cysteine, reduced apoptosis of SLC3. Several studies revealed that PA, which effectively maintained the redox state and also prevented the loss of intracellular antioxidants, antagonized the apoptotic cell death. Taken together, oxidative stress caused by decreased supply of PA might account for erythroid apoptosis due to R-PK deficiency.

Quantification of Intracellular Metabolites in Escherichia coli K12 Using Liquid Chromatographic-Electrospray Ionization Tandem Mass Spectrometric Techniques

The quantitative comprehension of microbial metabolic networks is a prerequisite for an efficient rational strain improvement (“metabolic engineering”). It is therefore necessary to accurately determine the concentration of a large number of reactants (i.e., metabolites, nucleotides, cofactors) in order to understand “in vivo” reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates and nucleotides in Escherichia coli K12 using a perchloric acid extraction and an LC-ESI-MS method was achieved. Intracellular metabolites (e.g., glucose 6-phosphate, fructose 1,6-bisphosphate, 6-phospho gluconate, acetyl-CoA, adenine nucleotides) were quantified under defined (glucose-limited steady-state) growth conditions. The method was verified by comparing the intracellular metabolite concentrations measured via LC-ESI-MS with enzymatic determinations. It is thus possible to identify and quantify more than 15 intracellular metabolites in parallel with a minimal amount of sample volume.

Molecular physiology of sugar catabolism in Lactococcus lactis IL1403.

The metabolic characteristics of Lactococcus lactis IL1403 were examined on two different growth media with respect to the physiological response to two sugars, glucose and galactose. Analysis of specific metabolic rates indicated that despite significant variations in the rates of both growth and sugar consumption, homolactic fermentation was maintained for all cultures due to the low concentration of either pyruvate-formate lyase or alcohol dehydrogenase. When the ionophore monensin was added to the medium, flux through glycolysis was not increased, suggesting a catabolic flux limitation, which, with the low intracellular concentrations of glycolytic intermediates and high in vivo glycolytic enzyme capacities, may be at the level of sugar transport. To assess transcription, a novel DNA macroarray technology employed RNA labeled in vitro with digoxigenin and detection of hybrids with an alkaline phosphatase-antidigoxigenin conjugate. This method showed that several genes of glycolysis were expressed to higher levels on glucose and that the genes of the mixed-acid pathway were expressed to higher levels on galactose. When rates of enzyme synthesis are compared to transcript concentrations, it can be deduced that some translational regulation occurs with threefold-higher translational efficiency in cells grown on glucose.

Metabolic Analysis of Glutamate Production by Corynebacterium glutamicum

The dynamic behavior of the metabolism of Corynebacterium glutamicum during l-glutamic acid fermentation, was evaluated by quantitative analysis of the evolution of intracellular metabolites and key enzyme concentrations. Glutamate production was induced by an increase of the temperature and a final concentration of 80g/l was attained. During the production phase, various other compounds, notably lactate, trehalose, and DHA were secreted to the medium. Intracellular metabolites analysis showed important variations of glycolytic intermediates and NADH, NAD coenzymes levels throughout the production phase. Two phenomena occur during the production phase which potentially provoke a decrease in the glutamate yield: Both the intracellular concentrations of glycolytic intermediates and the NADH/NAD ratio increase significantly during the period in which the overall metabolic rates decline. This correlates with the decrease in glutamate yield due in part to the production of lactate and also to the period of the fermentation in which growth no longer occurred.

Coupling physiology and gene regulation in bacteria: the phosphotransferase sugar uptake system delivers the signals.

In many bacteria a crucial link between metabolism and regulation of catabolic genes is based on the phosphotransferase sugar uptake system (PTS). We summarize the mechanisms of the signaling pathways originating from PTS and leading to regulation of transcription. A protein domain, called PTS regulation domain (PRD), is linked to many antiterminators and transcriptional activators and regulates their activity depending on its state of phosphorylation. Two sites can be phosphorylated in most PRDs: HPr-dependent modification at one site leads to activation while enzyme II dependent phosphorylation of the other site renders it inactive. In addition, PTS components are used to generate cofactors for regulators of transcription. The paradigm is the enzyme II dependent activity of adenylate cyclase determining the cyclic AMP level in Escherichia coli and thereby the activity of the catabolite activator protein. In many gram-positive bacteria catabolite repression is mediated by the catabolite control protein CcpA, which requires HPr Ser-46 phosphate as a cofactor to regulate transcription of catabolic genes. HPr Ser-46 phosphate is produced by HPr kinase, the activity of which is under metabolic control via the concentrations of glycolytic intermediates. These recent results establish a multifaceted regulatory role for PTS in addition to its well-established function in active sugar uptake.