Robustness of MetaNet graph models: predicting control of urea production in humans

Urea production in human liver was described by a MetaNet graph, a flowchart-like representation of metabolic pathways that includes parameters for the kinetic constants of the constituent enzymes. Formal operations on the graph facilitate the identification of ligand-binding equilibria that participate in feedback regulation in the network of biochemical reactions. The state of the biochemical network is specified by the concentrations of the intermediates. At any particular time, the influence of an identified locus of regulation is proportional to the respective fractional saturation of the corresponding binding site. Enzymes that make or consume the feedback chemicals share in the control of the strength of the feedback signal in proportion to their fractional saturation. This model predicts control of urea production by the processes that deliver amino groups to the urea cycle enzymes more than by the cycle enzymes themselves. Mitochondrial membrane transport processes are important for transmission of information through the network, but irreversible enzymes and processes far from equilibrium control the strength of the feedback signal. Systematic variation of the parameter values by amounts comparable to the expected variability of their measured values indicated a high probability of invariance in the identities of the predicted control points. The properties of the model are consistent with those of error-tolerant scale-free networks. These results demonstrate the robustness of a MetaNet model's predictions with respect to uncertainties in the values of its parameters.     

Topological analysis of metabolic control

A topological approach is presented for the analysis of control and regulation in metabolic pathways. In this approach, the control structure of a metabolic pathway is represented by a weighted directed graph. From an inspection of the topology of the graph, the control coefficients of the enzymes are evaluated in a heuristic manner in terms of the enzyme elasticities. The major advantage of the topological approach is that it provides a visual framework for (1) calculating the control coefficients of the enzymes, (2) analyzing the cause-effect relationships of the individual enzymes, (3) assessing the relative importance of the enzymes in metabolic regulation, and (4) simplifying the structure of a given pathway, from a regulatory viewpoint. Results are obtained for (a) an unbranched pathway in the absence of feedback the feedforward regulation and (b) an unbranched pathway with feedback inhibition. Our formulation is based on the metabolic control theory of Kacser and Burns (1973) and Heinrich and Rapoport (1974).     

Identification of regulatory properties of metabolic networks by graph theoretical modeling

An earlier graph theoretical model of metabolic and gene-expression networks has been modified and extended to include the effect of electrical potentials on binding constants, representation of uncatalyzed processes, and treatment of parallel reactions catalyzed by a single enzyme. Formal operations on the graph, which are facilitated by a set of standardized guidelines, identify the feedback signals in the network and rank them according to their influence. The technique was applied to a model of glycolysis in ascites tumor cells in the absence and presence of 12.5 mM exogenous glucose. Feedback regulation was widely distributed and mostly due to binding of adenine nucleotide cofactors to the enzymes of the network. The major changes in feedback regulation on adding glucose is the relief of inhibition of hexokinase and phosphofructokinase and the activation of pyruvate kinase. We conclude that regulation of tumor cell glycolysis is not restricted to hexokinase or to (Na+,K+)-ATPase as was previously suggested by others.     

Glycogen metabolizing enzymes in brain

Three enzymes, glycogen phosphorylase, glycogen synthase, and phosphoglucomutase were evaluated in subcellular fractions and in brain regions. Also the development of each of these enzymes was evaluated in whole brain homogenates. Each enzyme increased during the first three weeks of post partum in a manner that is similar to the development of glycolytic enzymes during this period. The specific activity of each enzyme in various subcellular fractions indicated that the enzymes were primarily soluble. Also unlike the glycolytic enzyme phosphoglycerate kinase, the glycogen metabolizing enzymes had a lower specific activity in synaptosomes than in particle free supernatant fractions of homogenates. Regarding regional distribution small (less than twofold) but significant differences were seen between different brain areas. An inverse relationship between the glycogen metabolizing enzymes and hexokinase was observed, that is, regions highest in glycogen synthase and glycogen phosphorylase were lowest in hexokinase and regions highest in hexokinase were lowest in the glycogen metabolizing enzymes.     

Regulation of yeast glycogen phosphorylase by the cyclin-dependent protein kinase Pho85p

Yeast accumulate glycogen in response to nutrient limitation. The key enzymes of glycogen synthesis and degradation, glycogen synthase, and phosphorylase, are regulated by reversible phosphorylation. Phosphorylation inactivates glycogen synthase but activates phosphorylase. The kinases and phosphatases that control glycogen synthase are well characterized whilst the enzymes modifying phosphorylase are poorly defined. Here, we show that the cyclin-dependent protein kinase, Pho85p, which we have previously found to regulate glycogen synthase also controls the phosphorylation state of phosphorylase.     

Lysosomal glycogen storage induced by Acarbose, a 1,4-alpha-glucosidase inhibitor.

The 1,4-alpha-glucosidase inhibitor. Acarbose, when injected intraperitoneally disturbs liver lysosome metabolism, causing distinct and persistent inhibition of the enzymes and acute disturbances of lysosomal glycogen metabolism. A feedback control mechanism appears to operate, affecting cytosolic carbohydrate metabolism. A model is suggested for the adult form of lysosomal storage disease. The biochemical effects closely resemble those occurring in glycogenosis type II (Pompe's disease), and these have been confirmed by electron microscopy.     

Bond graph simulation of skeletal muscle glucose metabolism.

A mathematical model of skeletal muscle glucose metabolism is presented. This model resulted from the application of thermodynamics combined with the dynamics of an energy storage capability. The physiological system consists of the insulin modulated, carrier-mediated transport of glucose into skeletal muscle, the biochemical reactions of the Embden-Meyerhof pathway, the cyclic 3′,5′ adenosine monophosphate-dependent protein kinase controlled synthesis/degradation of glycogen stores, and the diffusion of lactate from muscle. The metabolic system is defined and synthesized by the construction of the energy flow bond graph.The bond graph model was evaluated by simulating the system response over a 2 min period to step increases in extracellular epinephrine concentration. The simulated response of the metabolites and modulated enzymes corresponds, qualitatively and quantitatively, with in vivo measurements published in the literature.     

An enzymic fluorometric micro method for determination of glycoen

A sensitive and rapid micro determination of glycogen in biological samples has been described. The method is fluoro-enzymic and is based on the conversion of glycogen to 6-phosphogluconate with the enzymes amylo-α-1,4-α-1,6-glucosidase, hexokinase, and glucose-6-phosphate dehydrogenase. The increase in NADPH is measured fluorometrically. As little as 200 ng of glycogen can be determined.     

Phosphorylation-dependent translocation of glycogen synthase to a novel structure during glycogen resynthesis

Glycogen metabolism has been the subject of extensive research, but the mechanisms by which it is regulated are still not fully understood. It is well accepted that the rate-limiting enzymes in glycogenesis and glycogenolysis are glycogen synthase (GS) and glycogen phosphorylase (GPh), respectively. Both enzymes are regulated by reversible phosphorylation and by allosteric effectors. However, evidence in the literature indicates that changes in muscle GS and GPh intracellular distribution may constitute a new regulatory mechanism of glycogen metabolism. Already in the 1960s, it was proposed that glycogen was present in dynamic cellular organelles that were termed glycosomas but no such cellular entities have ever been demonstrated. The aim of this study was to characterize muscle GS and GPh intracellular distribution and to identify possible translocation processes of both enzymes. Using in situ stimulation of rabbit tibialis anterior muscle, we show GS and GPh intracellular redistribution at the beginning of glycogen resynthesis after contraction-induced glycogen depletion. We identify a new "player," a new intracellular compartment involved in skeletal muscle glycogen metabolism. They are spherical structures that were not present in basal muscle, and we present evidence that indicate that they are products of actin cytoskeleton remodeling. Furthermore, for the first time, we show a phosphorylation-dependent intracellular distribution of GS. Here, we present evidence of a new regulatory mechanism of skeletal muscle glycogen metabolism based on glycogen enzyme intracellular compartmentalization.     

Kinases and phosphatases of hepatic glycogen metabolism during fasted to refed transition in normal and streptozotocin-induced diabetic rats.

Normal and streptozotocin-induced diabetic rats were fasted for 24 hours and refed for 4 hours. Changes in the activities of glycogen metabolizing enzymes in liver were followed during this period. In normal rats, hepatic glycogen content increased gradually after the onset of food intake. The percent of active glycogen synthase increased to a peak value at 1h which coincided with a significant (P less than 0.02) increase in synthase phosphatase activity. Phosphorylase alpha and the percent of alpha increased significantly (P less than 0.01) after the meal which correlated with similar increases in cAMP-dependent protein kinase and phosphorylase kinase activities. Activation of enzymes involved in both synthesis and degradation of glycogen during fasted to refed transition indicate a probable substrate cycling. In diabetic livers, there was marked decrease in the activities of glycogen metabolizing enzymes and their levels did not alter significantly in response to the meal indicating a poor turnover of glycogen.     

Immunological characterization of Escherichia coli B glycogen synthase and branching enzyme and comparison with enzymes from other bacteria.

Escherichia coli B glycogen synthase and branching enzyme, although similar in amino acid composition, had no significant immunological cross-reactivity. The N-terminal sequences of the glycogen synthase were rich in hydrophobic residues, whereas branching enzyme had a higher content of acidic and basic residues. However, residues 21 to 28 of glycogen synthase and 7 to 14 of branching enzyme shared six of eight residues in common. Two fractions of branching enzyme, branching enzymes I and II, which can be isolated from E. coli B cell extracts, have been shown to be immunologically identical, suggesting that only one type of branching enzyme activity is present in E. coli B. Evidence has been obtained which indicates that E. coli B glycogen synthase and branching enzyme are antigenically very similar to glycogen synthases and branching enzymes from other enteric bacteria. No cross-reactivity with either enzyme was observed in cell extracts from photosynthetic bacteria.     

Glycogen and its metabolism

Glycogen is a branched polymer of glucose which serves as a reservoir of glucose units. The two largest deposits in mammals are in the liver and skeletal muscle but many cells are capable synthesizing glycogen. Its accumulation and utilization are under elaborate controls involving primarily covalent phosphorylation and allosteric ligand binding. Both muscle and liver glycogen reserves are important for whole body glucose metabolism and their replenishment is linked hormonally to nutritional status. Control differs between muscle and liver in part due to the existence of different tissue-specific isoforms at key steps. Control of synthesis is shared between transport into the muscle and the step catalyzed by glycogen synthase. Breakdown of liver glycogen, as part of blood glucose homeostasis, is also in response to nutritional cues. Muscle glycogen serves only to fuel muscular activity and its utilization is controlled by muscle contraction and by catecholamines. Though the number of enzymes directly involved in the metabolism of glycogen is quite small, many more proteins act indirectly in a regulatory capacity. Defects in the basic metabolizing enzymes lead to severe consequences whereas, with some exceptions, mutations in the regulatory proteins appear to cause a more subtle phenotypic change.     

Initiation of glycogen biosynthesis in Escherichia coli. Studies of the properties of the enzymes involved.

The properties of the enzymes involved in the initiation of glycogen biosynthesis in Escherichia coli were studied. It was found that the enzymic activities which transfer the glycosyl residues from UDPglucose or ADPglucose for the glucoprotein synthesis had differing stabilities upon storage at 4 degrees C. The small amount of glycogen and the saccharide firmly bound to the membrane preparation, were degraded during the storage period. The activity measured in fresh and in stored preparations gave different time dependence curves. The stored preparation had a lag period which could be due to the transfer of the first glucose units to the protein. Both UDPglucose and ADPglucose : protein glucosyltransferases were affected in different ways by detergents. Based on the results presented, it may be concluded that both enzymatic activities are due to different enzymes. Furthermore, both enzymatic activities are different from that which transfers glucose from ADPglucose to glycogen. The following mechanism for the de novo synthesis is suggested. Glycogen in E. coli could be initiated by two different enzymes which transfer glucose to a protein acceptor either from UDPglucose or ADPglucose. Once the saccharide linked to the protein has reached a certain size it is almost exclusively enlarged by another ADPglucose-dependent enzyme. The participation of branching enzyme will produce a polysaccharide with the characteristics of glycogen.     

Comparative characterization of human and rat liver glycogen synthase.

Glycogen synthase from human liver was studied, and its properties were compared with those of rat liver glycogen synthase. The rat and human liver glycogen synthases were similar in their pH profile, in their kinetic constants for the substrate UDP-glucose and the activator glucose 6-phosphate, and in their elution profiles from Q-Sepharose. The apparent molecular weight of the human synthase subunit was 80,000-85,000 by gel electrophoresis, which is similar to that of the rat enzyme. In addition, antibodies to rat liver glycogen synthase recognized human liver glycogen synthase, indicating that the enzymes of these two species share antigenic determinants. However, there were significant differences between the two enzymes. In particular, the total activity of the human enzyme was higher than that of the rat. The human enzyme, purified to near homogeneity, had a specific activity of 40 U/mg protein compared with 20 U/mg protein for the rat enzyme. The active forms of the rat enzyme had greater thermal stability than those of the human enzyme, but the human enzyme was more stable on storage in various buffers. Last, amino acid analysis indicated differences between the enzymes of the two species. Since glycogen synthase is an important enzyme in liver glycogen synthesis, the characterization of this enzyme in the human will help provide insight regarding human liver glycogen synthesis.     

The actions of cyclic AMP on biosynthetic processes are mediated indirectly by cyclic AMP-dependent protein kinase.

Adrenalin and glucagon inhibit glycogen, fatty acid and cholesterol synthesis by elevation of cyclic AMP, activation of cyclic AMP-dependent protein kinase and increased phosphorylation of the rate-limiting enzymes of these pathways. Here, we review recent evidence which indicates that inhibition of these biosynthetic pathways in muscle, adipose tissue and liver is much more indirect than has previously been supposed. In particular, cyclic AMP-dependent protein kinase does not appear to inhibit glycogen synthase, acetyl-CoA carboxylase and HMG-CoA reductase by phosphorylating them directly. It appears to achieve the same end result by inactivation of the protein phosphatases which dephosphorylate these regulatory enzymes in vivo, although this has only been established definitively in the case of glycogen synthesis.     

Relationship of Glycolytic Intermediates, Glycolytic Enzymes, and Ammonia to Glycogen Metabolism During Sporulation in the Yeast Saccharomyces cerevisiae

To identify the factors which control glycogen synthesis in Saccharomyces cerevisiae, we have studied the regulation of glycogen metabolism during sporulation, since in vivo glycogen has been reported to undergo significant changes in concentration during this process. We examined the concentration of a number of key glycolytic intermediates and enzymes in strains that sporulate at different rates and those that are deficient in sporulation. There were no significant changes found in the adenylate energy charge or cyclic AMP levels throughout sporulation. Although significant alterations occurred in the levels of glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, phosphoenolpyruvate, and ATP during sporulation, only the fourfold increase in fructose-1,6-bisphosphate appeared to correlate with glycogen synthesis in all of the strains examined. Only limited changes occurred in the level of a number of glycolytic and gluconeogenic enzymes which were examined during this process. Intracellular glucose content underwent a dramatic 30- to 40-fold increase in sporulating cells. Comparison of strains with different rates of sporulation demonstrated that this increase in glucose content coincides with the time of glycogen degradation in each strain. Both the increase in glucose content and the degradation of accumulated glycogen were not observed in nonsporulating alpha/alpha strains, or in cells incubated in NH(4) (+) supplemented sporulation medium. Although glucose appears to be the direct product of glycogen degradation, a 10-fold increase in a nonspecific alkaline phosphatase occurs at this time, which may be degrading phosphorylated sugars to glucose. All of the strains examined released extracellular glucose while suspended in acetate sporulation medium. It is concluded that most of the changes in the glycolytic pathway that occur during sporulation, with the exception of glycogen degradation and the concomitant increase in intracellular glucose pools, are a response to the transfer to sporulation medium and are independent of sporulation-specific processes. Inhibition of sporulation with ammonium ions resulted in a different pattern of change in all of the glycolytic intermediates examined, including a twofold increase in cyclic AMP levels. Ammonia did not interfere with glycogen synthesis, but prevented sporulation-specific glycogen degradation. The levels of the glycolytic enzymes examined were not affected by ammonia.     

The histochemical evaluation of the glycogen storage diseases. A review of techniques and their limitations

Synopsis Histochemical techniques have been applied to biopsies of liver and muscle in cases of glycogen storage disease in order to demonstrate the presence of glycogen and the various enzymes which are involved in the degradation and synthesis of glycogen. This review of techniques is based on the examination of tissues from a group of patients with glycogen storage disease, and the results have been correlated with biochemical assays on the same tissues. It is possible to differentiate between the various forms of glycogen storage disease with the exception of Types III and VI which, due to the vagaries of the phosphorylase reaction, have very similar staining characteristics. The techniques have also been applied successfully to blood films from which considerable information can be obtained.     

Initiation of glycogen biosynthesis in Escherichia coli studies of the properties of the enzymes involved

The properties of the enzymes involved in the initiation of glycogen biosynthesis in Escherichia coli were studied.It was found that the enzymic activities which transfer the glycosyl residues from UDPglucose or ADPglucose for the glucoprotein synthesis had differing stabilities upon storage at 4°C.The small amount of glycogen and the saccharide firmly bound to the membrane preparation, were degraded during the storage period.The activity measured in fresh and in stored preparations gave different time dependence curves. The stored preparation had a lag period which could be due to the transfer of the first glucose units to the protein.Both UDPglucose and ADPglucose: protein glucosyltransferases were affected in different ways by detergents.Based on the results presented, it may be concluded that both enzymatic activities are due to different enzymes. Furthermore, both enzymatic activities are different from that which transfers glucose from ADPglucose to glycogen.The following mechanism for the de novo synthesis is suggested. Glycogen in E. coli could be initiated by two different enzymes which transfer glucose to a protein acceptor either from UDPglucose or ADPglucose. Once the saccharide linked to the protein has reached a certain size it is almost exclusively enlarged by another ADPglucose-dependent enzyme. The participation of branching enzyme will produce a polysaccharide with the characteristics of glycogen.     

Regulation of glycogen synthesis in human skeletal muscle: does cellular glycogen control glycogen synthase phosphatase activity?

Contrary to the accepted feedback control mechanism of glycogen biosynthesis in skeletal muscle, evidence is presented here leading to the conclusion that glycogen does not control the activity of glycogen synthase phosphatase in intact human skeletal muscle tissue.     

Isolation of a polydisperse high-molecular-weight glycogen from rat liver

A new method is described for the isolation of glycogen from rat liver using centrifugation, gentle heating, and gel chromatography. The prepared polysaccharide was judged by both sucrose density gradient centrifugation and the absorbance spectrum of an I₂-glycogen complex to be highly branched, polydisperse, and of an unusually high molecular weight upon comparison to other glycogens. Using adult fasted rats, this glycogen was shown to be better than high-molecular-weight cold water-ethanol extracted glycogen for the binding of glycogen metabolizing enzymes. Further, the addition of 0.5% ($\text{w}\text{v}$) of the glycogen to a crude liver extract from newborn rats facilitated the isolation of an almost 700-fold purified glycogen synthase with 40% recovery. It is suggested that this glycogen could also be used to study the role of enzyme binding in the regulation of carbohydrate metabolism.