Changes in fructose-2,6-bisphosphate levels after glucose loading of starved rats

Fructose-2,6-bisphosphate levels in freeze-clamped livers of starved rats were 0.5 nmol/g liver. Oral administration of 1 g glucose per kg body weight to starved rats increased glycogen levels from 4 mg/g liver to 13.5 mg/g in 2 hr but did not significantly alter fructose-2,6-bisphosphate levels. The low level of this effector is consistent with an active gluconeogenic process and the results support the hypothesis that carbon atoms for glycogen synthesis can be derived from 3-carbon precursors via this pathway, even in the presence of glucose.     

Antihyperglycemic effect of fraxetin on hepatic key enzymes of carbohydrate metabolism in streptozotocin-induced diabetic rats

Epidemiological studies have demonstrated that the diabetes mellitus is a serious health burden for both governments and healthcare providers. The present study was hypothesized to evaluate the antihyperglycemic potential of fraxetin by determining the activities of key enzymes of carbohydrate metabolism in streptozotocin (STZ) – induced diabetic rats. Diabetes was induced in male albino Wistar rats by intraperitoneal administration of STZ (40 mg/kg b.w). Fraxetin was administered to diabetic rats intra gastrically at 20, 40, 80 mg/kg b.w for 30 days. The dose 80 mg/kg b.w, significantly reduced the levels of blood glucose and glycosylated hemoglobin (HbA1c) and increased plasma insulin level. The altered activities of the key enzymes of carbohydrate metabolism such as glucokinase, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, fructose-1,6-bisphosphatase and hepatic enzymes (aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP)) in the liver tissues of diabetic rats were significantly reverted to near normal levels by the administration of fraxetin. Further, fraxetin administration to diabetic rats improved body weight and hepatic glycogen content demonstrated its antihyperglycemic potential. The present findings suggest that fraxetin may be useful in the treatment of diabetes even though clinical studies to evaluate this possibility may be warranted.     

Effect of pterostilbene on hepatic key enzymes of glucose metabolism in streptozotocin- and nicotinamide-induced diabetic rats

The purpose of this study was to investigate the effect of pterostilbene and its effect on key enzymes of glucose metabolism. Diabetic rats were orally administered with pterostilbene (10, 20, 40 mg/kg) for 2, 4 and 6 weeks on glucose was determined. Administration of pterostilbene at 40 mg/kg significantly decreases plasma glucose. Based on these data, the higher dose, 40 mg/kg pterostilbene, was selected for further evaluation. Oral administration of pterostilbene for 6 weeks on glucose, insulin levels and hepatic enzymes in normal and streptozotocin (STZ)-nicotinamide-induced diabetic rats. A significant decrease in glucose and significant increase in plasma insulin levels were observed in normal and diabetic rats treated with pterostilbene. Treatment with pterostilbene resulted in a significant reduction of glycosylated hemoglobin and an increase in total hemoglobin level. The activities of the hepatic enzymes such as hexokinase was significantly increased whereas glucose-6-phosphatase, fructose-1,6-bisphosphatase were significantly decreased by the administration of pterostilbene in diabetic rats. A comparison was made between the action of pterostilbene and the antidiabetic drug--metformin.     

Efficacy of coumarin on hepatic key enzymes of glucose metabolism in chemical induced type 2 diabetic rats

The study was undertaken to evaluate the antidiabetic effect of coumarin on carbohydrate metabolic key enzymes in control and streptozotocin (STZ)-nicotinamide (NA)-induced diabetic rats. On oral administration of coumarin at a dose of 100 mg/kg body weight per day to diabetic rats for 45 days; resulted in a significant reduction in the levels of plasma glucose, glycosylated hemoglobin (HbA₁c) and increase in the levels of insulin and hemoglobin. Administration of coumarin caused a significant increase in the levels of glycolytic enzyme (hexokinase) and hepatic shunt enzyme (glucose-6-phophate dehydrogenase) whereas significant decrease in the levels of gluconeogenic enzymes (glucose-6-phosphatase and fructose-1,6-bisphosphatase) in diabetic treated rats. Furthermore, protection against body weight loss of diabetic animals also observed. This study indicates that the administration of coumarin to diabetic rats resulted in alterations in the metabolism of glucose with subsequent reduction in plasma glucose levels.     

Fermentative glycolysis with purified Escherichia coli enzymes for in vitro ATP production and evaluating an engineered enzyme

Each of the twelve enzymes for glycolytic fermentation, eleven from Escherichia coli and one from Saccharomyces cerevisiae, have been over-expressed in E. coli and purified with His-tags. Simple assays have been developed for each enzyme and they have been assembled for fermentation of glucose to ethanol. Phosphorus-31 NMR revealed that this in vitro reaction accumulates fructose 1,6-bisphosphate while recycling the cofactors NAD⁺ and ATP. This reaction represents a defined ATP-regeneration system that can be tailored to suit in vitro biochemical reactions such as cell-free protein synthesis. The enzyme from S. cerevisiae, pyruvate decarboxylase 1 (Pdc1; EC 4.1.1.1), was identified as one of the major ‘flux controlling’ enzymes for the reaction and was replaced with an evolved version of Pdc1 that has over 20-fold greater activity under glycolysis reaction conditions. This substitution was only beneficial when the ratio of glycolytic enzymes was adjusted to suit greater Pdc1 activity.     

Thiazolidine-2-carboxylate derivatives formed from glyoxylate and L-cysteine or L-cysteinylglycine as possible physiological substrates for D-aspartate oxidase

Adducts of glyoxylate with L-cysteine or L-cysteinylglycine were found to be excellent substrates at low concentrations for beef kidney D-aspartate oxidase. Evidence is presented that cis-thiazolidine-2,4-dicarboxylate and its glycine amide are the actual substrates, and that both are converted in the enzymic reaction to 4-substituted thiazoline-2-carboxylates. The results imply that these thiazolidine derivatives are the likely physiological reactants for mammalian D-aspartate oxidase.     

Studies of regulatory metabolism in Moniezia expansa: Glutamate,and the absence of the γ-aminobutyrate pathway

Moniezia expansa takes up radioactive glutamate from an isotonic medium, and radiocarbon appears primarily in α-ketoglutarate and succinate. Glutamate-oxaloacetate and glutamate-pyruvate transaminase activities were present in subcellular preparations; however, the enzymes of the γ-aminobutyrate pathway were absent. Further attempts to obtain indirect evidence for the operation of this pathway failed, and the metabolism of glutamate appears to take place via a preliminary transamination to α-ketoglutarate, followed by oxidation to succinate.     

A new regulatory principle for in vivo biochemistry: Pleiotropic low affinity regulation by the adenine nucleotides – Illustrated for the glycolytic enzymes of Saccharomyces cerevisiae

Enzymology tends to focus on highly specific effects of substrates, allosteric modifiers, and products occurring at low concentrations, because these are most informative about the enzyme’s catalytic mechanism. We hypothesized that at relatively high in vivo concentrations, important molecular monitors of the state of living cells, such as ATP, affect multiple enzymes of the former and that these interactions have gone unnoticed in enzymology.     

A path analysis of the causal relationships between red cell glycolytic intermediates, ADP and ATP in sickle cell anemia

The statistical relationships among the glycolytic intermediates (GI)) of the Embden-Meyerhof pathway, adenine nucleotides (ANs) and various hematological measures were estimated for 34 sickle cell anemia patients. Heterogeneity in linear and quadratic regressions of hemoglobin and hematocrit, both singly and jointly, on the GI and AN variables implied 1) that any single formula to standardize optical density measures of the GIs and ANs on a per gram hemoglobin or per liter cell water basis would not uniformly remove hemoglobin and hematocrit effects: 2) that ignoring significant hematological effects could bias the estimates of correlation among GIs and ANs; and 3) that hemoglobin and hematocrit measures do not reflect the same source of variability. The correlations among the GIs and ANs, after adjustment for hematological variability, were analyzed by path analysis to determine which of five proposed path models for cause and effect relationships were compatible with the data. AMP had a greater influence on ADP (coefficient of determination (CD) = 23%) than all the GIs together, while G6P and ADP influenced ATP variability the most (CD = 33% and 12%). The contributions of unknown factors to ADP and ATP variability were large for all models (CD = 56--77%) possibly due to stress of sickle cell disease. The path model with AMP and the four GIs (G6P, F6P, FDP, DHAP) influencing ADP variation, and the same GIs and ADP influencing ATP was the model most compatible with the data.     

A comparative analysis of kinetic models of erythrocyte glycolysis

Since the 1970s, with Heinrich as a pioneer in the field, numerous kinetic models of erythrocyte glycolysis have been constructed. A functional comparison of eight of these models indicates that the production of ATP and GSH in the red blood cell is largely controlled by the demand reactions. The rate characteristics for the supply and demand blocks indicate a good homeostatic control of ATP and GSH concentrations at different work loads for the pathway, while the production rates of ATP and GSH can be adjusted as needed by the demand reactions.     

Mapping photoautotrophic metabolism with isotopically nonstationary (13)C flux analysis

Understanding in vivo regulation of photoautotrophic metabolism is important for identifying strategies to improve photosynthetic efficiency or re-route carbon fluxes to desirable end products. We have developed an approach to reconstruct comprehensive flux maps of photoautotrophic metabolism by computational analysis of dynamic isotope labeling measurements and have applied it to determine metabolic pathway fluxes in the cyanobacterium Synechocystis sp. PCC6803. Comparison to a theoretically predicted flux map revealed inefficiencies in photosynthesis due to oxidative pentose phosphate pathway and malic enzyme activity, despite negligible photorespiration. This approach has potential to fill important gaps in our understanding of how carbon and energy flows are systemically regulated in cyanobacteria, plants, and algae.     

Strain engineering for microbial production of value-added chemicals and fuels from glycerol

While the widespread reliance on fossil fuels is driven by their low cost and relative abundance, this fossil-based economy has been deemed unsustainable and, therefore, the adoption of sustainable and environmentally compatible energy sources is on the horizon. Biorefinery is an emerging approach that integrates metabolic engineering, synthetic biology, and systems biology principles for the development of whole-cell catalytic platforms for biomanufacturing. Due to the high degree of reduction and low cost, glycerol, either refined or crude, has been recognized as an ideal feedstock for the production of value-added biologicals, though microbial dissimilation of glycerol sometimes can be difficult particularly under anaerobic conditions. While strain development for glycerol biorefinery is widely reported in the literature, few, if any, commercialized bioprocesses have been developed as a result, such that engineering of glycerol metabolism in microbial hosts remains an untapped opportunity in biomanufacturing. Here we review the recent progress made in engineering microbial hosts for the production of biofuels, diols, organic acids, biopolymers, and specialty chemicals from glycerol. We begin with a broad outline of the major pathways for fermentative and respiratory glycerol dissimilation and key end metabolites, and then focus our analysis on four key genera of bacteria known to naturally dissimilate glycerol, i.e. Klebsiella, Citrobacter, Clostridium, and Lactobacillus, in addition to Escherichia coli, and systematically review the progress made toward engineering these microorganisms for glycerol biorefinery. We also identify the major biotechnological and bioprocessing advantages and disadvantages of each genus, and bottlenecks limiting the production of target metabolites from glycerol in engineered strains. Our analysis culminates in the development of potential strategies to overcome the current technical limitations identified for commonly employed strains, with an outlook on the suitability of different hosts for the production of key metabolites and avenues for their future development into biomanufacturing platforms.     

Systems biology of the metabolic network regulated by the Akt pathway

Cancer has been proposed as an example of systems biology disease or network disease. Accordingly, tumor cells differ from their normal counterparts more in terms of intracellular network dynamics than single markers. Here we shall focus on a recently recognized hallmark of cancer, the deregulation of cellular energetics. The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been confirmed as an essential step toward cell transformation. We will consider how the effects of Akt activation are connected with cell metabolism; more precisely, we will review existing metabolic models and discuss the current knowledge available to construct a kinetic model of the most relevant metabolic processes regulated by the PI3K/Akt pathway. The model will enable a systems biology approach to predict the metabolic targets that may inhibit cell growth under hyper activation of Akt.     

The pro-tumorigenic effects of metabolic alterations in glioblastoma including brain tumor initiating cells

De-regulated cellular energetics is an emerging hallmark of cancer with alterations to glycolysis, oxidative phosphorylation, the pentose phosphate pathway, lipid oxidation and synthesis and amino acid metabolism. Understanding and targeting of metabolic reprogramming in cancers may yield new treatment options, but metabolic heterogeneity and plasticity complicate this strategy. One highly heterogeneous cancer for which current treatments ultimately fail is the deadly brain tumor glioblastoma. Therapeutic resistance, within glioblastoma and other solid tumors, is thought to be linked to subsets of tumor initiating cells, also known as cancer stem cells. Recent profiling of glioblastoma and brain tumor initiating cells reveals changes in metabolism, as compiled here, that may be more broadly applicable. We will summarize the profound role for metabolism in tumor progression and therapeutic resistance and discuss current approaches to target glioma metabolism to improve standard of care.     

In Saccharomyces cerevisiae,withdrawal of the carbon source results in detachment of glycolytic enzymes from the cytoskeleton and in actin reorganization

Metabolons are dynamic associations of enzymes catalyzing consecutive reactions within a given pathway. Association results in enzyme stabilization and increased metabolic efficiency. Metabolons may use cytoskeletal elements, membranes and membrane proteins as scaffolds. The effects of glucose withdrawal on a putative glycolytic metabolon/F-actin system were evaluated in three Saccharomyces cerevisiae strains: a WT and two different obligate fermentative (OxPhos-deficient) strains, which obtained most ATP from glycolysis. Carbon source withdrawal led to inhibition of fermentation, decrease in ATP concentration and dissociation of glycolytic enzymes from F-actin. Depending on the strain, inactivation/reactivation transitions of fermentation took place in seconds. In addition, when ATP was very low, green fluorescent protein-labeled F-actin reorganized from highly dynamic patches to large, non-motile actin bodies containing proteins and enzymes. Glucose addition restored fermentation and cytoskeleton dynamics, suggesting that in addition to ATP concentration, at least in one of the tested strains, metabolon assembly/disassembly is a factor in the control of the rate of fermentation.     

Quantitative evaluation of respiration induced metabolic oscillations in erythrocytes

The changes in the partial pressures of oxygen and carbon dioxide (P_O2 and P_CO2) during blood circulation alter erythrocyte metabolism, hereby causing flux changes between oxygenated and deoxygenated blood. In the study we have modeled this effect by extending the comprehensive kinetic model by Mulquiney and Kuchel [P.J. Mulquiney, and P.W. Kuchel. Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: equations and parameter refinement, Biochem. J. 1999, 342, 581–596.] with a kinetic model of hemoglobin oxy-/deoxygenation transition based on an oxygen dissociation model developed by Dash and Bassingthwaighte [R. Dash, and J. Bassingthwaighte. Blood HbO₂ and HbCO₂ dissociation curves at varied O₂, CO₂, pH, 2,3-DPG and temperature levels, Ann. Biomed. Eng., 2004, 32(12), 1676–1693.]. The system has been studied during transitions from the arterial to the venous phases by simply forcing P_O2 and P_CO2 to follow the physiological values of venous and arterial blood. The investigations show that the system passively follows a limit cycle driven by the forced oscillations of P_O2 and is thus inadequately described solely by steady state consideration. The metabolic system exhibits a broad distribution of time scales. Relaxations of modes with hemoglobin and Mg²⁺ binding reactions are very fast, while modes involving glycolytic, membrane transport and 2,3-BPG shunt reactions are much slower. Incomplete slow mode relaxations during the 60 s period of the forced transitions cause significant overshoots of important fluxes and metabolite concentrations – notably ATP, 2,3-BPG, and Mg²⁺. The overshoot phenomenon arises in consequence of a periodical forcing and is likely to be widespread in nature – warranting a special consideration for relevant systems.