A proteome-integrated, carbon source dependent genetic regulatory network in Saccharomyces cerevisiae.

Integrated regulatory networks can be powerful tools to examine and test properties of cellular systems, such as modelling environmental effects on the molecular bioeconomy, where protein levels are altered in response to changes in growth conditions. Although extensive regulatory pathways and protein interaction data sets exist which represent such networks, few have formally considered quantitative proteomics data to validate and extend them. We generate and consider such data here using a label-free proteomics strategy to quantify alterations in protein abundance for S. cerevisiae when grown on minimal media using glucose, galactose, maltose and trehalose as sole carbon sources. Using a high quality-controlled subset of proteins observed to be differentially abundant, we constructed a proteome-informed network, comprising 1850 transcription factor interactions and 37 chaperone interactions, which defines the major changes in the cellular proteome when growing under different carbon sources. Analysis of the differentially abundant proteins involved in the regulatory network pointed to their significant roles in specific metabolic pathways and function, including glucose homeostasis, amino acid biosynthesis, and carbohydrate metabolic process. We noted strong statistical enrichment in the differentially abundant proteome of targets of known transcription factors associated with stress responses and altered carbon metabolism. This shows how such integrated analysis can lend further experimental support to annotated regulatory interactions, since the proteomic changes capture both magnitude and direction of gene expression change at the level of the affected proteins. Overall this study highlights the power of quantitative proteomics to help define regulatory systems pertinent to environmental conditions.

Metabolite Depletion Affects Flux Profiling of Cell Lines.

Quantifying the rate of consumption and release of metabolites (i.e., flux profiling) has become integral to the study of cancer. The fluxes as well as the growth of the cells may be affected by metabolite depletion during cultivation.

Development of a minimal growth medium for Lactobacillus plantarum.

AIM: A medium with minimal requirements for the growth of Lactobacillus plantarum WCFS was developed. The composition of the minimal medium was compared to a genome-scale metabolic model of L. plantarum. METHODS AND RESULTS: By repetitive single omission experiments, two minimal media were developed: PMM5 (true minimal medium) and PMM7 [a pseudominimal medium, supporting proper biomass formation of 350 mg l(-1) dry weight (DW)]. The specific growth rate of L. plantarum on PMM7 was found to be 50% and 63% lower when compared to growth on established growth media (chemically defined medium and MRS, respectively). Using a genome-scale metabolic model of L. plantarum, it was predicted that PMM5 and PMM7 would not support the growth of L. plantarum. This is because the biosynthesis of para-aminobenzoic acid (pABA) was predicted to be essential for growth. The discrepancy in simulated growth and experimental growth on PMM7 was further investigated for pABA; a molecule which plays an important role in folate production. The growth performance and folate production were determined on PMM7 in the presence and absence of pABA. It was found that a 12,000-fold reduction in folate pools exerted no influence on formation of biomass or growth rate of L. plantarum cultures when grown in the absence of pABA. CONCLUSION: Largely reduced folate production pools do not have an effect on the growth of L. plantarum, showing that L. plantarum makes folate in a large excess. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments illustrate the importance of combining genome-scale metabolic models with growth experiments on minimal media.

Leptin deficiency per se dictates body composition and insulin action in ob/ob mice.

Obese humans are often insulin- and leptin resistant. Since leptin can affect glucose metabolism, it is conceivable that a lack of leptin signal transduction contributes to insulin resistance. It remains unclear whether leptin affects glucose metabolism via peripheral and/or central mechanistic routes. In the present study, we aimed: (i) to determine the relative contributions of lack of leptin signal transduction and adiposity to insulin resistance and (ii) to establish the impact of central leptin action on glucose metabolism. To address the first point, ob/ob mice were subjected to severe calorie restriction, so that their body weight became similar to that of wild-type mice. Insulin sensitivity was measured in obese ob/ob, lean (food restricted) ob/ob and lean, weight-matched wild-type mice. To address the second point, leptin (or vehicle) was i.c.v. infused to the lateral cerebral ventricle of ob/ob mice and insulin sensitivity was determined. Hyperinsulinaemic euglyceamic clamps were used to quantify insulin sensitivity. Food restriction barely affected body composition, although it profoundly curtailed body weight. Insulin suppressed hepatic glucose production (HGP) to a greater extent in lean ob/ob than in obese ob/ob mice, but its impact remained considerably less than in wild-type mice (% suppression: 11.8 +/- 8.9 versus 1.3 +/- 1.1 versus 56.6 +/- 13.0%/nmol, for lean, obese ob/ob and wild-type mice, respectively; P < 0.05). The insulin-mediated glucose disposal (GD) of lean ob/ob mice was also in between that of obese ob/ob and wild-type mice (37.5 +/- 21.4 versus 25.1 +/- 14.6 versus 59.6 +/- 17.3 mumol/min/kg/nmol of insulin, respectively; P < 0.05 wild-type versus obese ob/ob mice). Leptin infusion acutely enhanced both hepatic insulin sensitivity (insulin-induced inhibition of HGP) and insulin-mediated GD (9.1 +/- 2.4 versus 5.0 +/- 2.7%/nmol of insulin, and 25.6 +/- 5.6 versus 13.6 +/- 4.8 mumol/min/kg/nmol of insulin, respectively; P < 0.05 for both comparisons) in ob/ob mice. Both a lack of leptin signals and adiposity may contribute to insulin resistance in obese individuals. Diminution of central leptin signalling can critically affect glucose metabolism in these individuals.

Stimulation of the in vivo production of very low density lipoproteins by apolipoprotein E is independent of the presence of the low density lipoprotein receptor.

Apolipoprotein (apo) E stimulates the secretion of very low density lipoproteins (VLDLs) by an as yet unknown mechanism. Recently, a working mechanism for apoE was proposed (Twisk, J., Gillian-Daniel, D. L., Tebon, A., Wang, L., Barrett, P. H., and Attie, A. D. (2000) J. Clin. Invest. 105, 521-532) in which apoE prevents the inhibitory action of the low density lipoprotein receptor (LDLr) by binding to it. We have first tested whether this newly described effect of the LDLr on VLDL secretion, obtained in vitro, is also observed in vivo. In LDLr knockout mice (LDLr-/-), the production of VLDL triglycerides and apoB was 30% higher than that in controls. Also the ratio of apoB100:apoB48 secretion was increased in the LDLr-/- mice. The composition of nascent VLDL was similar in both strains. To test whether the action of apoE depends on the presence of the LDLr, VLDL production was measured in LDLr-/- and apoE-/- LDLr-/- mice. Deletion of apoE on a LDLr-/- background still caused a 50% decrease of VLDL triglycerides and apoB production. The composition of nascent VLDL was again similar for both strains. We conclude that the effect of apoE on hepatic VLDL production is independent of the presence of the LDLr.

Mice expressing only the mutant APOE3Leiden gene show impaired VLDL secretion.

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 micromol/kg per hour, respectively). The presence of a single mouse apoe allele increased VLDL-TG secretion in APOE3Leiden/apoe+/- mice (121+/-43 micromol/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.

Control of glycolytic dynamics by hexose transport in Saccharomyces cerevisiae.

It is becoming accepted that steady-state fluxes are not necessarily controlled by single rate-limiting steps. This leaves open the issue whether cellular dynamics are controlled by single pacemaker enzymes, as has often been proposed. This paper shows that yeast sugar transport has substantial but not complete control of the frequency of glycolytic oscillations. Addition of maltose, a competitive inhibitor of glucose transport, reduced both average glucose consumption flux and frequency of glycolytic oscillations. Assuming a single kinetic component and a symmetrical carrier, a frequency control coefficient of between 0.4 and 0.6 and an average-flux control coefficient of between 0.6 and 0.9 were calculated for hexose transport activity. In a second approach, mannose was used as the carbon and free-energy source, and the dependencies on the extracellular mannose concentration of the transport activity, of the frequency of oscillations, and of the average flux were compared. In this case the frequency control coefficient and the average-flux control coefficient of hexose transport activity amounted to 0.7 and 0.9, respectively. From these results, we conclude that 1) transport is highly important for the dynamics of glycolysis, 2) most but not all control resides in glucose transport, and 3) there should at least be one step other than transport with substantial control.

A functional genomics strategy that uses metabolome data to reveal the phenotype of silent mutations.

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.

LDL receptor deficiency unmasks altered VLDL triglyceride metabolism in VLDL receptor transgenic and knockout mice.

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.

Can yeast glycolysis be understood in terms of in vitro kinetics of the constituent enzymes? Testing biochemistry.

This paper examines whether the in vivo behavior of yeast glycolysis can be understood in terms of the in vitro kinetic properties of the constituent enzymes. In nongrowing, anaerobic, compressed Saccharomyces cerevisiae the values of the kinetic parameters of most glycolytic enzymes were determined. For the other enzymes appropriate literature values were collected. By inserting these values into a kinetic model for glycolysis, fluxes and metabolites were calculated. Under the same conditions fluxes and metabolite levels were measured. In our first model, branch reactions were ignored. This model failed to reach the stable steady state that was observed in the experimental flux measurements. Introduction of branches towards trehalose, glycogen, glycerol and succinate did allow such a steady state. The predictions of this branched model were compared with the empirical behavior. Half of the enzymes matched their predicted flux in vivo within a factor of 2. For the other enzymes it was calculated what deviation between in vivo and in vitro kinetic characteristics could explain the discrepancy between in vitro rate and in vivo flux.

'Slave' metabolites and enzymes. A rapid way of delineating metabolic control.

Although control of fluxes and concentrations tends to be distributed rather than confined to a single rate-limiting enzyme, the extent of control can differ widely between enzymes in a metabolic network. In some cases, there are enzymes that lack control completely. This paper identifies one surprising origin of such lack of control: If, in a metabolic system, there is a metabolite that affects the catalytic rate of only one enzyme, the corresponding enzyme cannot control any metabolic variable other than the concentration of that metabolite. We call such enzymes 'slave enzymes', and the corresponding metabolites 'slave metabolites'. Implications of the existence of slave enzymes for the control properties of enzymes further down the metabolic pathway are discussed and examined for the glycolytic pathway of yeast. Inadvertent assumptions in metabolic models may cause the latter incorrectly to calculate absence of metabolic control. The phenomenon of slave enzymes may well be important in enhancing metabolic signal transduction.

Compartmentation protects trypanosomes from the dangerous design of glycolysis.

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.

The danger of metabolic pathways with turbo design.

Many catabolic pathways begin with an ATP-requiring activation step, after which further metabolism yields a surplus of ATP. Such a 'turbo' principle is useful but also contains an inherent risk. This is illustrated by a detailed kinetic analysis of a paradoxical Saccharomyces cerevisiae mutant; the mutant fails to grow on glucose because of overactive initial enzymes of glycolysis, but is defective only in an enzyme (trehalose 6-phosphate synthase) that appears to have little relevance to glycolysis. The ubiquity of pathways that possess an initial activation step, suggests that there might be many more genes that, when deleted, cause rather paradoxical regulation phenotypes (i.e. growth defects caused by enhanced utilization of growth substrate).

Intracellular glucose concentration in derepressed yeast cells consuming glucose is high enough to reduce the glucose transport rate by 50%.

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.

Control analysis of glycolytic oscillations.

The principles involved in the control of the frequency of sustained metabolic oscillations are developed in the context of glycolytic oscillations in Saccharomyces cerevisiae. To this purpose, an existing mathematical model that describes the experimentally obtained oscillations was simplified to a core model. Frequency, relative phase, average concentrations and amplitudes of the oscillations were well approximated by writing the two remaining metabolic variables of the core model (representing [ATP] and [hexose]) as harmonic functions of time and by requiring them to fulfill the differential equations. The extent to which an enzyme (-conglomerate) controls the frequency in a sustained oscillation is defined as the log-log derivative of that frequency with respect to enzyme activity. In both the full model and the core model this control of frequency and the control over the average concentrations proved to be distributed over the enzymes. We identified a summation theorem, stating that the sum of such control coefficients over all processes equals unity for frequency and zero for the average concentrations.

Synchronized heat flux oscillations in yeast cell populations.

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.