Metabolites are the end products of cellular regulatory processes, and their levels can be regarded as the ultimate response of biological systems to genetic or environmental changes. In parallel to the terms ‘transcriptome’ and ‘proteome’, the set of metabolites synthesized by a biological system constitute its ‘metabolome’. Yet, unlike other functional genomics approaches, the unbiased simultaneous identification and quantification of plant metabolomes has been largely neglected. Until recently, most analyses were restricted to profiling selected classes of compounds, or to fingerprinting metabolic changes without sufficient analytical resolution to determine metabolite levels and identities individually. As a prerequisite for metabolomic analysis, careful consideration of the methods employed for tissue extraction, sample preparation, data acquisition, and data mining must be taken. In this review, the differences among metabolite target analysis, metabolite profiling, and metabolic fingerprinting are clarified, and terms are defined. Current approaches are examined, and potential applications are summarized with a special emphasis on data mining and mathematical modelling of metabolism.

https://link.springer.com/content/pdf/10.1023%2FA%3A1013713905833.pdf

Metabolomics - the link between genotypes and phenotypes

Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I.

Recent studies indicate that phosphatidylinositol 3-kinase is essential in the regulation of many processes dependent on membrane flow. Autophagy is a complex pathway in which cell material, including proteins, can be degraded. Membrane flow plays a pivotal role in this process. To find out whether phosphatidylinositol 3-kinase is also required for autophagy, we tested the effects on autophagy of two structurally unrelated phosphatidylinositol 3-kinase inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenylchromone (LY294002). The addition of low concentrations of each of these inhibitors to incubations of hepatocytes in the absence of amino acids resulted in a strong inhibition of proteolysis. The antiproteolytic effect of wortmannin (IC50 30 nM) and LY294002 (IC50 10 mu M) was accompanied by inhibition of autophagic sequestration and not by an increase in lysosomal pH or a decrease in intracellular ATP. No further inhibition of proteolysis by the two compounds was observed when autophagy was already maximally inhibited by high concentrations of amino acids. 3-Methyladenine, which is commonly used as a specific inhibitor of autophagic sequestration, was an inhibitor of phosphatidylinositol 3-kinase, thus providing a target for its action. It is proposed that phosphatidylinositol 3-kinase activity is required for autophagy. 3-Methyladenine inhibits autophagy by inhibition of this enzyme.

/pdf/the-phosphatidylinositol-3-kinase-inhibitors-wortmannin-and-36s35z8s91.pdf

The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes

As the details of the chemical transformations in metabolism have become increasingly clear, and the enzymes catalysing many of the reactions have been characterized, it is understandable that biochemists should want to explain at the molecular level the metabolic homeostasis observed at the physiological level. How are the rates of synthesis and degradation of metabolites kept in close balance over a very wide range of external conditions without catastrophic rises or falls in the metabolite concentrations? The discoveries of feedback inhibition, co-operativity and covalent modification in enzymes, and of mechanisms for the control of enzyme synthesis and degradation, have disclosed a repertoire of molecular effects that potentially alter the fluxes in metabolic pathways. With such a range of effects to choose from, it is not surprising that disputes arise over explanations for the changes in flux through particular pathways under given circumstances. Since the explanations are usually verbal and qualitative, discrimination between different explanations, or assessment of their adequacy, is difficult. More recently, several groups have attempted theoretical analysis of the potential of these different molecular mechanisms to contribute to the control of metabolic flux. Since these theories can be given a mathematical formulation, they can be used in combination with appropriate experimental measurements to provide quantitative explanations and, potentially, predictions. The theories have often been controversial. Matters at issue have included the extent to which it is feasible to perform experiments to obtain the necessary data, the adequacy of the theories for making useful predictions, and the degree to which the quantitative measures of the theories do actually capture the relevant aspects of regulation and control. To some extent, this is a matter of semantics; a mathematical theory is more explicit about its underlying assumptions and the meaning of its statements, but regulation and control are two terms that have been used without strict adherence to any agreed definition in many different contexts (as noted in [1]). It is therefore inevitable that some will find that the use of these terms in the context of a mathematical theory places a narrower construction on them than they would like. These theories all include a form of sensitivity analysis; that is, the magnitude of the effect of some small change in a parameter (such as an enzyme activity) on a metabolic system property (such as the flux or the concentration of a metabolite) is mathematically related to the properties of the components of the system. Sensitivity analysis is widely used for analogous problems in other fields, including economics, ecology [2], engineering [3] and chemical kinetics [4-6]. Its application in biochemistry was pioneered by Higgins [7], but three variants subsequently arose: Metabolic Control Analysis, Biochemical Systems Theory and Crabtree and Newsholme's 'flux-oriented' theory (the term used in [8]). It is not possible to give a succinct account of the differences between the approaches, which is a controversial area [9-18] even though the underlying mathematics is equivalent to a considerable extent. One area of difference is the choice of the type of parameter that is changed for the determination of sensitivities. In Metabolic Control Analysis, enzyme concentration (or activity) is usually chosen; the response to an external modifier of a metabolic pathway is derived from the resulting sensitivities. In Biochemical Systems Theory [19-25], the primary parameters for the sensitivities are the 'rate constants' for synthesis and degradation of metabolite pools. Savageau has given many reasons for this choice of parameter; Cornish-Bowden has articulated some of the problems with it [15]. Although using these 'rate constants' simplifies the analysis procedures within Biochemical Systems Theory, there is not a one-to-one relationship between them and the enzymes of the system, which can create a slight complication in determining the sensitivity to variation of an enzyme activity. Savageau's theory is part of an integrated system for stability analysis and simulation, in addition to sensitivity analysis. Crabtree and Newsholme's theory [8,10,26-30] is intermediate between the two others, and the primary sensitivities are to an external modifier (a hypothetical one if necessary), but its mathematical development is less rigorous. In this review, I shall concentrate on Metabolic Control Analysis. This is because, apart from considerations of space, approximately two-thirds of the literature citations of theories of metabolic regulation in the past 5 years have been to Metabolic Control Analysis. This may relate to perceived ease of use, which has been compared using the different approaches on the same set of experimental results [31]. In the following review, I will not give a complete derivation and description of the basic concepts of Metabolic Control Analysis; clear accounts can be found in previous articles and reviews [9,32-39]. Instead I will try to indicate areas of disagreement, the scope of the basic theory and where it has been modified or extended, and recent approaches to experimental applications.

/pdf/metabolic-control-analysis-a-survey-of-its-theoretical-and-2f9dujatvt.pdf

Metabolic control analysis: a survey of its theoretical and experimental development.

Phosphorylation of Ribosomal Protein S6 Is Inhibitory for Autophagy in Isolated Rat Hepatocytes

We have used control analysis to quantify the distribution of control in the gluconeogenic pathway in liver cells from starved rats. Lactate and pyruvate were used as gluconeogenic substrates. The flux control coefficients of the various enzymes in the gluconeogenic pathway were calculated from the elasticity coefficients of the enzymes towards their substrates and products and the fluxes through the different branches in the pathway. The elasticity coefficients were either calculated from gamma/Keq. ratios (where gamma is the mass-action ratio and Keq. is the equilibrium constant) and enzyme-kinetic data or measured experimentally. It is concluded that the gluconeogenic enzyme pyruvate carboxylase and the glycolytic enzyme pyruvate kinase play a central role in control of gluconeogenesis. If pyruvate kinase is inactive, gluconeogenic flux from lactate is largely controlled by pyruvate carboxylase. The low elasticity coefficient of pyruvate carboxylase towards its product oxaloacetate minimizes control by steps in the gluconeogenic pathway located after pyruvate carboxylase. This situation occurs when maximal gluconeogenic flux is required, i.e. in the presence of glucagon. In the absence of the hormone, when pyruvate kinase is active, control of gluconeogenesis is distributed among many steps, including pyruvate carboxylase, pyruvate kinase, fructose-1,6-bisphosphatase and also steps outside the classic gluconeogenic pathway such as the adenine-nucleotide translocator.

/pdf/control-of-gluconeogenesis-in-rat-liver-cells-flux-control-449yyfucxs.pdf

Control of gluconeogenesis in rat liver cells. Flux control coefficients of the enzymes in the gluconeogenic pathway in the absence and presence of glucagon.

The catabolism of alanine in isolated rat liver cells was studied using a perifusion system in order to titrate the cells with different concentrations of alanine.



The Concentrations of the components of the alanine aminotransferase reaction in the cytosolic and mitochondrial compartments were measured after digitonin fractionation of samples of the cell suspension taken during each steady state. Comparison of the mass-action ratios with the equilibrium constant indicated that the cytosolic enzyme is poised towards pyruvate formation at all concentrations of alanine used, whereas the mitochondrial enzyme is always poised towards alanine formation. The calculated flux through cytosolic alanine aminotransferase, using literature data for the kinetic parameters of the enzyme and experimental data for the concentration of metabolites, was in close agreement with the measured carbon flux. It is concluded that the transamination step in alanine catabolism occurs in the cytosol.



The concentration of the components of the glutamate dehydrogenase and 3-hydroxybutyrate dehydrogenase reactions in the mitochondrial compartment during each steady state were also measured. The ΔG of the combined reactions did not change appreciably on increasing the extracellular alanine concentration to 0.6 mM. At higher alanine concentrations ΔG became slightly more negative. As the extracellular Concentration of alanine was increased, the concentration of glutamate in both the cytosolic and the mitochondrial compartments increased. However, the cytosolic alanine concentration was always lower than that in the extracellular compartment even at the highest concentration of alanine (6 mM) infused. When increasing concentrations of pyruvate were infused together with a fixed concentration of alanine (0.6 mM), the cytosolic concentration of alanine increased to levels even higher than that in the perifusate. However, there was no significant effect in the rate of formation of nitrogenous products.



It is concluded that the transport of alanine across the plasma membrane is the only rate-controlling step in alanine catabolism under the conditions used.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1982.tb05851.x

Intracellular Compartment ation and Control of Alanine Metabolism in Rat Liver Parenchymal Cells

Control of gluconeogenesis in rat liver cells. I. Kinetics of the individual enzymes and the effect of glucagon

Control of gluconeogenesis from lactate was studied by titrating rat liver cells with lactate and pyruvate in a ratio of 1 O : l in a perifusion system. At different steady states of glucose formation, the concentration of key gluconeogenic intermediates was measured and plotted against gluconeogenic flux (Jglucoee). Complete saturation was observed only in the plot relating JglUcto the extracellular pyruvate concentration. Measurement of pyruvate distribution in the cell showed that the mitochondrial pyruvate translocator operates close to equilibrium at high lactate and pyruvate concentrations. It can therefore be concluded that pyruvate carboxylase limits maximal gluconeogenic flux. Addition of glucagon did not cause a shift in the plots relating Jglueose to glucose 6-phosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, and phosphoenolpyruvate. It can thus be concluded that glucagon does not affect the kinetic parameters of the enzymes involved in the conversion of phosphoenolpyruvate to glucose. Addition of glucagon led to a shift in the curves relating Jglueose to the concentration of cytosolic oxalacetate and extracellular pyruvate. The shift in the curve relating JgluCto oxalacetate is due to glucagon-induced inhibition of pyruvate kinase. The stimulation of gluconeogenesis by glucagon can be accounted for almost completely by inhibition of pyruvate kinase. There was almost no stimulation by glucagon of pyruvate carboxylation. In the absence of glucagon, control on gluconeogenesis from lactate is distributed among different steps including pyruvate carboxylase and pyruvate kinase. Assuming that in the presence of glucagon all pyruvate kinase flux is inhibited, the control of gluconeogenesis in the presence of the hormone is confined exclusively to pyruvate carboxylase.

Richard C. Vervoorn

Papers

Control of gluconeogenesis in rat liver cells. Flux control coefficients of the enzymes in the gluconeogenic pathway in the absence and presence of glucagon.

Intracellular Compartment ation and Control of Alanine Metabolism in Rat Liver Parenchymal Cells

Control of gluconeogenesis in rat liver cells. I. Kinetics of the individual enzymes and the effect of glucagon

Control of gluconeogenesis in rat liver cells

Control of mitochondrial respiration.