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A large proportion of the 6,000 genes present in the genome of Saccharomyces cerevisiae, and of those sequenced in other organisms, encode proteins of unknown function. Many of these genes are "silent, " that is, they show no overt phenotype, in terms of growth rate or other fluxes, when they are deleted from the genome. We demonstrate how the intracellular concentrations of metabolites can reveal phenotypes for proteins active in metabolic regulation. Quantification of the change of several metabolite concentrations relative to the concentration change of one selected metabolite can reveal the site of action, in the metabolic network, of a silent gene. In the same way, comprehensive analyses of metabolite concentrations in mutants, providing "metabolic snapshots," can reveal functions when snapshots from strains deleted for unstudied genes are compared to those deleted for known genes. This approach to functional analysis, using comparative metabolomics, we call FANCY-an abbreviation for functional analysis by co-responses in yeast.

Apolipoprotein E (apoE)-deficient mice develop hepatic steatosis and show impaired very low density lipoprotein (VLDL)-triglyceride (TG) secretion. These effects are normalized on the introduction of the human APOE3 gene. To assess whether this apoE effect is isoform specific, we studied hepatic lipid metabolism in mice expressing either APOE3 or the mutant APOE3Leiden on apoe −/− or apoe+ /− backgrounds. The transgenes were expressed mainly in periportal hepatocytes, as revealed by in situ hybridization. Mice expressing APOE3Leiden, on the apoe −/− and apoe+ /− backgrounds, had fatty livers, which were absent in APOE3/ apoe −/− mice. APOE3Leiden/ apoe −/− mice showed a strongly reduced VLDL-TG secretion compared with APOE3/ apoe −/− mice (48±14 versus 82±10 μmol/kg per hour, respectively). The presence of a single mouse apoe allele increased VLDL-TG secretion in APOE3Leiden/ apoe+ /− mice (121±43 μ mol/kg per hour) compared with APOE3Leiden/ apoe −/− mice. These results show that APOE3Leiden does not prevent development of a fatty liver and does not normalize VLDL-TG secretion in mice with an apoE-deficient background. The presence of a single mouse apoe allele is sufficient to normalize the APOE3Leiden-associated reduction of VLDL-TG secretion but does not prevent steatosis. We conclude that apoE-mediated stimulation of VLDL secretion is isoform specific.

Unlike in other organisms, in trypanosomes and other Kinetoplastida the larger part of glycolysis takes place in a specialized organelle, called the glycosome. At present it is impossible to remove the glycosome without changing much of the rest of the cell. It would seem impossible, therefore, to assess the metabolic consequences of this compartmentation. Therefore, we here develop a computer experimentation approach, which we call computational cell biology. A validated molecular kinetic computer replica was built of glycolysis in the parasite Trypanosoma brucei. Removing the glycosome membrane in that replica had little effect on the steady-state flux, which argues against the prevalent speculation that glycosomes serve to increase flux by concentrating the enzymes. Removal of the membrane did cause ( i ) the sugar phosphates to rise to unphysiologically high levels, which must have pathological effects, and ( ii ) a failure to recover from glucose deprivation. We explain these effects on the basis of the biochemical organization of the glycosome. We conclude ( i ) that the glycosome protects trypanosomes from the negative side effects of the “turbo” structure of glycolysis and ( ii ) that computer experimentation based on solid molecular data is a powerful tool to address questions that are not, or not yet, accessible to experimentation.

Much of biochemistry finds its roots in the study of metabolism. Substantial progress came when it was discovered that important metabolic processes, such as fermentation of glucose to lactate by erythrocytes or to ethanol by yeast, consisted of series of apparently independent chemical reactions. The independence of these reactions was established by purifying protein fractions that were each capable of uniquely catalysing one of the reactions. Likewise membrane-dependent biological free-energy transduction was reduced to the action of two proton pumps, i.e. one linked to the electron-transfer chain in the mitochondrial or bacterial inner membrane, the other coupled to ATP hydrolysis (Mitchell, 1979). Accordingly, biochemistry and molecular biology assimilated the paradigm that each of the physiological processes of the living cell could be understood in terms of a series of independent biochemical reactions (Fig. 1). Although some processes are reluctant to be reduced in this manner this is usually believed to be a matter of practice rather than of principle.

Publisher Summary This chapter discusses metabolic control analysis (MCA) as a tool in the elucidation of the function of novel genes. MCA is an attempt to understand quantitatively the global behavior of complex systems on the basis of the local properties of the components—the enzymes. In the past decade, substantial progress has been made to extend the MCA theory to more and more complex systems, such as large systems, group transfer pathways, channeling, and many more. The chapter explains the basic concepts and theorems of MCA because they may be applied to the functional analysis of novel genes. It explains MCA with respect to its implications for the elucidation of the functions of novel genes. All the experiments demonstrated in the chapter are done in silico using a simple kinetic model that mimics functional analysis experiments. A simple example is used in the chapter to illustrate the principles of MCA: the regulation of phosphofructokinase (PFK) by fructose, 2,6-bisphosphate (F26bP). The definitions and concepts of metabolic control analysis are also discussed in the chapter.

Microcalorimetry was adapted to the study of glycolytic oscillations in suspensions of intact yeast cells. A correction procedure was developed for the distortion of the amplitude and phase of the heat signal, caused by the slow response of the calorimeter. This made it possible to observe oscillations in the heat production rate with a period of less than 1 min, and a relative amplitude of 5-10%. By simultaneously measuring the heat flux and concentrations of glycolytic metabolites, and by comparing acetaldehyde-induced phase shifts of the heat flux oscillations with those of NADH oscillations, the heat flux was found to be 100 degrees out of phase with glucose 6-phosphate, 80 degrees out of phase with fructose 1, 6-bisphosphate, and in phase with NADH. The flux measurement made possible by microcalorimetry allowed the recognition of (i) changes in metabolic capacity that may affect glycolytic dynamics, (ii) implications of glucose carrier kinetics for glycolytic dynamics and (iii) the continued requirement for an acetaldehyde trapping agent for the oscillations.

Maintenance (let alone growth) of the highly ordered living cell is only possible through the continuous input of free energy. Coupling of energetically downhill processes (such as catabolic reactions) to uphill processes is essential to provide this free energy and is catalyzed by enzymes either directly or via "storage" in an intermediate high energy form, i.e., high ATP/ADP ratio or H+ ion gradient. Although maintenance of a sufficiently high ATP/ADP ratio is essential to overcome the thermodynamic burden of uphill processes, it is not clear to what degree enzymes that control this ratio also control cell physiology. Indeed, in the living cell homeostatic control mechanisms might exist for the free-energy transduction pathways so as to prevent perturbation of cellular function when the Gibbs energy supply is compromised. This presentation addresse the extent to which the intracellular ATP level is involved in the control of cell physiology, how the elaborate control of cell function may be analyzed theoretically and quantitatively, and if this can be utilized selectively to affect certain cell types.

In the glycolytic pathway, oscillations in metabolite concentrations occur in different organisms under different conditions1. In intact yeast cells, oscillations in glycolysis can be monitored by NAD(P)H fluorescence. For the induction of these oscillations, glucose is added to starved cells. After a steady state is reached, blocking respiration leads to oscillations in the NAD(P)H concentration with a frequency of 1.2 min-1 at a temperature of 25 °C. The damping of the oscillations depends on the state of growth before harvesting. Sustained oscillations can be observed when the cells are harvested at the transition from using glucose to ethanol as a substrate2. Furthermore there is evidence that the damping is also influenced by the cell concentration during the NAD(P)H measurement as first recognized by Pye3. This was interpreted as a consequence of intercellular communication4. In this paper we investigate the influence of the cell concentration on the duration of the oscillations and collect further evidence for intercellular communication.

The growth rate-dependent regulation of cell size, ribosomal content, and metabolic efficiency follows a common pattern in unicellular organisms: with increasing growth rates, cell size and ribosomal content increase and a shift to energetically inefficient metabolism takes place. The latter two phenomena are also observed in fast growing tumour cells and cell lines. These patterns suggest a fundamental principle of design. In biology such designs can often be understood as the result of the optimization of fitness. Here we show that in basic models of self-replicating systems these patterns are the consequence of maximizing the growth rate. Whereas most models of cellular growth consider a part of physiology, for instance only metabolism, the approach presented here integrates several subsystems to a complete self-replicating system. Such models can yield fundamentally different optimal strategies. In particular, it is shown how the shift in metabolic efficiency originates from a tradeoff between investments in enzyme synthesis and metabolic yields for alternative catabolic pathways. The models elucidate how the optimization of growth by natural selection shapes growth strategies. © 2009 EMBO and Macmillan Publishers Limited.

Background Thioredoxin (TRX) is a powerful disulfide oxido-reductase that catalyzes a wide spectrum of redox reactions in the cell. The aim of this study is to elucidate the role of the TRX system in the oxidative stress response in Lactobacillus plantarum WCFS1. Results We have identified the trxB1-encoded thioredoxin reductase (TR) as a key enzyme in the oxidative stress response of Lactobacillus plantarum WCFS1. Overexpression of the trxB1 gene resulted in a 3-fold higher TR activity in comparison to the wild-type strain. Subsequently, higher TR activity was associated with an increased resistance towards oxidative stress. We further determined the global transcriptional response to hydrogen peroxide stress in the trxB1-overexpression and wild-type strains grown in continuous cultures. Hydrogen peroxide stress and overproduction of TR collectively resulted in the up-regulation of 267 genes. Additionally, gene expression profiling showed significant differential expression of 27 genes in the trxB1-overexpression strain. Over expression of trxB1 was found to activate genes associated with DNA repair and stress mechanisms as well as genes associated with the activity of biosynthetic pathways for purine and sulfur-containing amino acids. A total of 16 genes showed a response to both TR overproduction and hydrogen peroxide stress. These genes are involved in the purine metabolism, energy metabolism (gapB) as well as in stress-response (groEL, npr2), and manganese transport (mntH2). Conclusion Based on our findings we propose that overproduction of the trxB1-encoded TR in L. plantarum improves tolerance towards oxidative stress. This response coincides with simultaneous induction of a group of 16 transcripts of genes. Within this group of genes, most are associated with oxidative stress response. The obtained crossover between datasets may explain the phenotype of the trxB1-overexpression strain, which appears to be prepared for encountering oxidative stress. This latter property can be used for engineering robustness towards oxidative stress in industrial strains of L. plantarum.

The very low density lipoprotein receptor (VLDLR) has been proposed to play a role in the delivery of fatty acids to peripheral tissues. However, despite reduced adipose tissue mass in VLDLR-deficient (VLDLR-/-) mice, this has been difficult to substantiate. In the present study, VLDLR-deficient and VLDLR-overexpressing (PVL) mice were cross-bred onto a low density lipoprotein receptor knockout (LDLR-/-) background to study the VLDLR under conditions of relatively high serum VLDL and triglyceride levels. Absence of the VLDLR resulted in a significant increase in serum triglyceride levels (1.9-fold) when mice were fed a high fat diet. In contrast, overexpression of the VLDLR resulted in a significant decrease in serum triglyceride levels (2.0-fold) under similar conditions. When kept on a chow diet, a period of prolonged fasting revealed a significant increase in serum triglyceride levels in VLDLR-/-; LDLR-/- mice (2.3-fold) as compared with LDLR-/- controls. This could not be attributed to altered apolipoprotein B and VLDL triglyceride production rates. Furthermore, no major differences in nascent VLDL triglyceride content were found between VLDLR-/-; LDLR-/- mice and LDLR-/- controls. However, the triglyceride content of circulating VLDL of VLDLR-/-; LDLR-/- mice (63%) was relatively high as compared with LDLR-/- controls (49%). These observations suggest that the VLDLR affects peripheral uptake of VLDL triglycerides. In conclusion, under conditions of LDLR deficiency in combination with high fat feeding or prolonged fasting, the effect of the VLDLR on VLDL triglyceride metabolism was revealed.—Tacken, P. J., B. Teusink, M. C. Jong, D. Harats, L. M. Havekes, K. Willems van Dijk, and M. H. Hofker. LDL receptor deficiency unmasks altered VLDL triglyceride metabolism in VLDL receptor transgenic and knockout mice. J. Lipid Res. 2000. 41: 2055–2062.

In Saccharomyces cerevisiae cells exhibiting high-affinity glucose transport, the glucose consumption rate at extracellular concentrations above 10 mM was only half of the zero trans -influx rate. To determine if this regulation of glucose transport might be a consequence of intracellular free glucose we developed a new method to measure intracellular glucose concentrations in cells metabolizing glucose, which compares glucose stereoisomers to correct for adhering glucose. The intracellular glucose concentration was 1.5 mM, much higher than in most earlier reports. We show that for the simplest model of a glucose carrier, this concentration is sufficient to reduce the glucose influx by 50%. We conclude that intracellular glucose is the most likely candidate for the observed regulation of glucose import and hence glycolysis. We discuss the possibility that intracellular glucose functions as a primary signal molecule in these cells.