Abstract: To what extent can modes of gene regulation be explained by systems-level properties of metabolic networks? Prior studies on co-regulation of metabolic genes have mainly focused on graph-theoretical features of metabolic networks and demonstrated a decreasing level of co-expression with increasing network distance, a naïve, but widely used, topological index. Others have suggested that static graph representations can poorly capture dynamic functional associations, e.g., in the form of dependence of metabolic fluxes across genes in the network. Here, we systematically tested the relative importance of metabolic flux coupling and network position on gene co-regulation, using a genome-scale metabolic model of Escherichia coli. After validating the computational method with empirical data on flux correlations, we confirm that genes coupled by their enzymatic fluxes not only show similar expression patterns, but also share transcriptional regulators and frequently reside in the same operon. In contrast, we demonstrate that network distance per se has relatively minor influence on gene co-regulation. Moreover, the type of flux coupling can explain refined properties of the regulatory network that are ignored by simple graph-theoretical indices. Our results underline the importance of studying functional states of cellular networks to define physiologically relevant associations between genes and should stimulate future developments of novel functional genomic tools.

Abstract: 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.

Abstract: ABSTRACT: 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.

Abstract: Lactic acid bacteria (LAB) have a long tradition of use in the food industry, and the number and diversity of their applications has increased considerably over the years. Traditionally, process optimization for these applications involved both strain selection and trial and error. More recently, metabolic engineering has emerged as a discipline that focuses on the rational improvement of industrially useful strains. In the post-genomic era, metabolic engineering increasingly benefits from systems biology, an approach that combines mathematical modelling techniques with functional-genomics data to build models for biological interpretation and--ultimately--prediction. In this review, the industrial applications of LAB are mapped onto available global, genome-scale metabolic modelling techniques to evaluate the extent to which functional genomics and systems biology can live up to their industrial promise.

Abstract: BACKGROUND: The genomic information of a species allows for the genome-scale reconstruction of its metabolic capacity. Such a metabolic reconstruction gives support to metabolic engineering, but also to integrative bioinformatics and visualization. Sequence-based automatic reconstructions require extensive manual curation, which can be very time-consuming. Therefore, we present a method to accelerate the time-consuming process of network reconstruction for a query species. The method exploits the availability of well-curated metabolic networks and uses high-resolution predictions of gene equivalency between species, allowing the transfer of gene-reaction associations from curated networks. RESULTS: We have evaluated the method using Lactococcus lactis IL1403, for which a genome-scale metabolic network was published recently. We recovered most of the gene-reaction associations (i.e. 74 - 85%) which are incorporated in the published network. Moreover, we predicted over 200 additional genes to be associated to reactions, including genes with unknown function, genes for transporters and genes with specific metabolic reactions, which are good candidates for an extension to the previously published network. In a comparison of our developed method with the well-established approach Pathologic, we predicted 186 additional genes to be associated to reactions. We also predicted a relatively high number of complete conserved protein complexes, which are derived from curated metabolic networks, illustrating the potential predictive power of our method for protein complexes. CONCLUSION: We show that our methodology can be applied to accelerate the reconstruction of genome-scale metabolic networks by taking optimal advantage of existing, manually curated networks. As orthology detection is the first step in the method, only the translated open reading frames (ORFs) of a newly sequenced genome are necessary to reconstruct a metabolic network. When more manually curated metabolic networks will become available in the near future, the usefulness of our method in network prediction is likely to increase.

Abstract: A genome-scale metabolic model of the lactic acid bacterium Lactobacillus plantarum WCFS1 was constructed based on genomic content and experimental data. The complete model includes 721 genes, 643 reactions, and 531 metabolites. Different stoichiometric modeling techniques were used for interpretation of complex fermentation data, as L. plantarum is adapted to nutrient-rich environments and only grows in media supplemented with vitamins and amino acids. (i) Based on experimental input and output fluxes, maximal ATP production was estimated and related to growth rate. (ii) Optimization of ATP production further identified amino acid catabolic pathways that were not previously associated with free-energy metabolism. (iii) Genome-scale elementary flux mode analysis identified 28 potential futile cycles. (iv) Flux variability analysis supplemented the elementary mode analysis in identifying parallel pathways, e.g. pathways with identical end products but different co-factor usage. Strongly increased flexibility in the metabolic network was observed when strict coupling between catabolic ATP production and anabolic consumption was relaxed. These results illustrate how a genome-scale metabolic model and associated constraint-based modeling techniques can be used to analyze the physiology of growth on a complex medium rather than a minimal salts medium. However, optimization of biomass formation using the Flux Balance Analysis approach, reported to successfully predict growth rate and by product formation in Escherichia coli and Saccharomyces cerevisiae, predicted too high biomass yields that were incompatible with the observed lactate production. The reason is that this approach assumes optimal efficiency of substrate to biomass conversion, and can therefore not predict the metabolically inefficient lactate formation.

Abstract: Dietary fatty acids have a profound impact on atherosclerosis, but mechanisms are not fully understood. We studied the effects of a saturated fat diet supplemented with fish oil, trans10,cis12 conjugated linoleic acid (CLA), or elaidic acid on lipid and glucose metabolism and liver protein levels of APOE*3 Leiden transgenic mice, a model for lipid metabolism and atherosclerosis. Fish oil lowered plasma and liver cholesterol and triglycerides, plasma free fatty acids, and glucose but increased plasma insulin. CLA lowered plasma cholesterol but increased plasma and liver triglycerides, plasma beta-hydroxybutyrate, and insulin. Elaidic acid lowered plasma and liver cholesterol. Proteomics identified significant regulation of 65 cytosolic and 8-membrane proteins. Many of these proteins were related to lipid and glucose metabolism, and to oxidative stress. Principal component analysis revealed that fish oil had a major impact on cytosolic proteins, and elaidic acid on membrane proteins. Correlation analysis between physiological and protein data revealed novel clusters of correlated variables, among which a metabolic syndrome cluster. The combination of proteomics and physiology gave new insights in mechanisms by which these dietary fatty acids regulate lipid metabolism and related pathways, for example, by altering protein levels of long-chain acyl-CoA thioester hydrolase and adipophilin in the liver.

Abstract: Hepatic VLDL and glucose production is enhanced in type 2 diabetes and associated with hepatic steatosis. Whether the derangements in hepatic metabolism are attributable to steatosis or to the increased availability of FA metabolites is not known. We used methyl palmoxirate (MP), an inhibitor of carnitine palmitoyl transferase I, to acutely inhibit hepatic FA oxidation and investigated whether the FAs were rerouted into VLDL secretion and whether this would affect hepatic glucose production. After an overnight fast, male APOE3*Leiden transgenic mice received an oral dose of 10 mg/kg MP. Administration of MP led to an 83% reduction in plasma beta-hydroxybutyrate (ketone body) levels compared with vehicle-treated mice (0.47 +/- 0.07 vs. 2.81 +/- 0.16 mmol/l, respectively; P < 0.01), indicative of impaired ketogenesis. Plasma FFA levels were increased by 32% and cholesterol and insulin levels were decreased by 17% and 50%, respectively, in MP-treated mice compared with controls. MP treatment led to a 30% increase in liver triglyceride (TG) content. Surprisingly, no effect on hepatic VLDL-TG production was observed between the groups at 8 h after MP administration. In addition, the capacity of insulin to suppress endogenous glucose production was unaffected in MP-treated mice compared with controls. In conclusion, acute inhibition of FA oxidation increases hepatic lipid content but does not stimulate hepatic VLDL secretion or reduce insulin sensitivity.

Abstract: Our aim was to study whether the absence of apolipoprotein (apo) C3, a strong inhibitor of lipoprotein lipase (LPL), accelerates the development of obesity and consequently insulin resistance. Apoc3(-/-) mice and wild-type littermates were fed a high-fat (46 energy %) diet for 20 weeks. After 20 weeks of high-fat feeding, apoc3(-/-) mice showed decreased plasma triglyceride levels (0.11 +/- 0.02 vs. 0.29 +/- 0.04 mmol, P < 0.05) and were more obese (42.8 +/- 3.2 vs. 35.2 +/- 3.3 g; P < 0.05) compared with wild-type littermates. This increase in body weight was entirely explained by increased body lipid mass (16.2 +/- 5.9 vs. 10.0 +/- 1.8 g; P < 0.05). LPL-dependent uptake of triglyceride-derived fatty acids by adipose tissue was significantly higher in apoc3(-/-) mice. LPL-independent uptake of albumin-bound fatty acids did not differ. It is interesting that whole-body insulin sensitivity using hyperinsulinemic-euglycemic clamps was decreased by 43% and that suppression of endogenous glucose production was decreased by 25% in apoc3(-/-) mice compared with control mice. Absence of apoC3, the natural LPL inhibitor, enhances fatty acid uptake from plasma triglycerides in adipose tissue, which leads to higher susceptibility to diet-induced obesity followed by more severe development of insulin resistance. Therefore, apoC3 is a potential target for treatment of obesity and insulin resistance.

Abstract: The biotechnology industry continuously explores new ways to improve the performance of microbial strains in fermentation processes. Recent focus has been on new genome-wide modelling approaches in functional genomics, which aim to take full advantage of genome sequence data, transcription profiling, proteomics and metabolite profiling for strain improvement. The integration of global metabolic models with genetic and regulatory models will be essential for the practice of metabolic engineering for strain improvement to move forward, simply because we cannot rely on our intuition to grasp the complexity of the biological systems involved.

Abstract: On the basis of the annotated genome we reconstructed the metabolic pathways of the lactic acid bacterium Lactobacillus plantarum WCFS1. After automatic reconstruction by the Pathologic tool of Pathway Tools (http://bioinformatics.ai.sri.com/ptools/), the resulting pathway-genome database, LacplantCyc, was manually curated extensively. The current database contains refinements to existing routes and new gram-positive bacterium-specific reactions that were not present in the MetaCyc database. These reactions include, for example, reactions related to cell wall biosynthesis, molybdopterin biosynthesis, and transport. At present, LacplantCyc includes 129 pathways and 704 predicted reactions involving some 670 chemical species and 710 enzymes. We tested vitamin and amino acid requirements of L. plantarum experimentally and compared the results with the pathways present in LacplantCyc. In the majority of cases (32 of 37 cases) the experimental results agreed with the final reconstruction. LacplantCyc is the most extensively curated pathway-genome database for gram-positive bacteria and is open to the microbiology community via the World Wide Web (www.lacplantcyc.nl). It can be used as a reference pathway-genome database for gram-positive microbes in general and lactic acid bacteria in particular.

Abstract:

Abstract: A key complication in comparative genomics for reliable gene function prediction is the existence of duplicated genes. To study the effect of gene duplication on function prediction, we analyze orthologs between pairs of genomes where in one genome the orthologous gene has duplicated after the speciation of the two genomes (i.e. inparalogs). For these duplicated genes we investigate whether the gene that is most similar on the sequence level is also the gene that has retained the ancestral gene-neighborhood. Although the majority of investigated cases show a consistent pattern between sequence similarity and gene-neighborhood conservation, a substantial fraction, 29-38%, is inconsistent. The observation of inconsistency is not the result of a chance outcome owing to a lack of divergence time between inparalogs, but rather it seems to be the result of a chance outcome caused by very similar rates of sequence evolution of both inparalogs relative to their ortholog. If one-to-one orthologous relationships are required, it is advisable to combine contextual information (i.e. gene-neighborhood in prokaryotes and co-expression in eukaryotes) with protein sequence information to predict the most probable functional equivalent ortholog in the presence of inparalogs.

Abstract: The prospect of understanding the relationship between the genome and the physiology of an organism is an important incentive to reconstruct metabolic networks. The first steps in the process can be automated and it does not take much effort to obtain an initial metabolic reconstruction from a genome sequence. However, such a reconstruction is certainly not flawless and correction of the many imperfections is laborious. It requires the combined analysis of the available information on protein sequence, phylogeny, gene-context and co-occurrence but is also aided by high-throughput experimental data. Simultaneously, the reconstructed network provides the opportunity to visualize the "omics" data within a relevant biological functional context and thus aids the interpretation of those data.

Abstract: BACKGROUND/AIMS: Apolipoprotein E (apoE)-deficient mice develop hepatic steatosis and secrete reduced levels of VLDL-TG. METHODS AND RESULTS: We examined the effects of apoE-deficiency on intracellular lipid homeostasis and secretion of triglycerides (TG). We show that intracellular TG turnover and activities of diacylglycerol acyltransferase (DGAT) and microsomal triglyceride transfer protein (MTP) are similar in Apoe(-/-) and wild type mice. In addition, apoB synthesis was not decreased in Apoe(-/-) cells. Thus, the accumulation of lipid in these cells is not attributable to perturbed TG turnover, apoB synthesis, and the activities of DGAT and MTP. Inhibition of MTP had a more profound impact on the secretion of VLDL-TG from wild type hepatocytes than Apoe(-/-) hepatocytes, indicating that MTP was more limiting for the production of VLDL-TG from wild type cells. In marked contrast to the MTP-deficient model of fatty liver, electron microscopy of lipid-stained liver sections of Apoe(-/-) mice revealed an accumulation of lipid in numerous small, putative ER-derived vesicles and in the cytosol. No abnormalities were observed in the Golgi of Apoe(-/-) mice. CONCLUSIONS: These results suggest that the removal of lipids from the early or intermediary compartments of the secretory pathway of hepatocytes is impaired in Apoe(-/-) mice.

Abstract: Lactic acid bacteria (LAB) have been widely used in food fermentations and, more recently, as probiotics in health-promoting food products. Genome sequencing and functional genomics studies of a variety of LAB are now rapidly providing insights into their diversity and evolution and revealing the molecular basis for important traits such as flavor formation, sugar metabolism, stress response, adaptation and interactions. Bioinformatics plays a key role in handling, integrating and analyzing the flood of 'omics' data being generated. Reconstruction of metabolic potential using bioinformatics tools and databases, followed by targeted experimental verification and exploration of the metabolic and regulatory network properties, are the present challenges that should lead to improved exploitation of these versatile food bacteria.

Abstract: The VLDL receptor (VLDLr) is involved in tissue delivery of VLDL-triglyceride (TG)-derived FFA by facilitating the expression of lipoprotein lipase (LPL). However, vldlr-/- mice do not show altered plasma lipoprotein levels, despite reduced LPL expression. Because LPL activity is crucial in postprandial lipid metabolism, we investigated whether the VLDLr plays a role in chylomicron clearance. Fed plasma TG levels of vldlr-/- mice were 2.5-fold increased compared with those of vldlr+/+ littermates (1.20 +/- 0.37 mM vs. 0.47 +/- 0.18 mM; P < 0.001). Strikingly, an intragastric fat load led to a 9-fold increased postprandial TG response in vldlr-/- compared with vldlr+/+ mice (226 +/- 188 mM/h vs. 25 +/- 11 mM/h; P < 0.05). Accordingly, the plasma clearance of [3H]TG-labeled protein-free chylomicron-mimicking emulsion particles was delayed in vldlr-/- compared with vldlr+/+ mice (half-life of 12.0 +/- 2.6 min vs. 5.5 +/- 0.9 min; P < 0.05), with a 60% decreased uptake of label into adipose tissue (P < 0.05). VLDLr deficiency did not affect the plasma half-life and adipose tissue uptake of albumin-complexed [14C]FFA, indicating that the VLDLr facilitates postprandial LPL-mediated TG hydrolysis rather than mediating FFA uptake. We conclude that the VLDLr plays a major role in the metabolism of postprandial lipoproteins by enhancing LPL-mediated TG hydrolysis.

Abstract: Familial dysbetalipoproteinemia associated with the apolipoprotein E2 (APOE2) genotype is a recessive disorder with low penetrance. We have investigated whether additional expression of full-length APOE3, APOE4, or a truncated variant of APOE4 (APOE4-202) can reduce APOE2- associated hyperlipidemia. This was achieved using adenovirus-mediated gene transfer to mice transgenic for human APOE2 and deficient for endogenous Apoe (APOE2.Apoe-/- mice). The hyperlipidemia of APOE2.Apoe-/- mice was readily aggravated by APOE3 and APOE4 overexpression. Only a very low dose of APOE4 adenovirus was capable of reducing the serum cholesterol and triglyceride (TG) levels. Expression of higher doses of APOE4 was associated with an increased VLDL-TG production rate and the accumulation of TG-rich VLDL in the circulation. In contrast, a high dose of adenovirus carrying APOE4-202 reduced both the cholesterol and TG levels in APOE2.Apoe-/- mice. Despite the absence of the C-terminal lipid-binding domain, APOE4-202 is apparently capable of binding to lipoproteins and mediating hepatic uptake. Moreover, overexpression of APOE4-202 in APOE2.Apoe-/- mice does not aggravate their hypertriglyceridemia. These results extend our previous analyses of APOE4-202 expression in Apoe-/- mice and demonstrate that apoE4-202 functions even in the presence of clearance-defective apoE2. Thus, apoE4-202 is a safe and efficient candidate for future therapeutic applications.

Abstract: There is controversy over the extent to which fatty acids (FAs) derived from plasma free FAs (FFAs) or from hydrolysis of plasma triglycerides (TGFAs) form communal or separate pools and what the contribution of each FA source is to cellular FA metabolism. Chylomicrons and lipid emulsions were labeled with [(3)H]triolein, injected into mice, and appearance in plasma of [(3)H]oleic acid was estimated, either through a steady-state approach or by compartmental modeling. [(14)C]oleic acid was included to trace plasma FFA. Eighty to 90% of triglyceride (TG) label was recovered in plasma, irrespective of tracer method or TG source. The contribution of TG lipolysis to total plasma FA turnover was 10-20%. After infusion of [(3)H]TG and [(14)C]FA, the retention of these labels varied substantially among liver, adipose tissue, and skeletal and heart muscle. Retention of TG label changed during fasting in the same direction as lipoprotein lipase (LPL) activity is regulated. We propose a model that reconciles the paradoxical 80-90% loss of TG label into plasma with LPL-directed differential uptake of TGFA in tissues. In this model, TGFAs mix locally at the capillaries with plasma FFAs, where they would lead to an increase in the local FA concentration, and hence, FA uptake. Our data indicate that a distinction between TG-derived FA and plasma FFA cannot be made.

Abstract: Hormone-sensitive lipase (HSL) is a major enzyme for triglyceride (TG) lipolysis in adipose tissue. In HSL-knockout mice, plasma free fatty acid and TG levels are low, associated with low liver TG content. Because a decreased hepatic insulin sensitivity has been reported to be associated with high liver TG levels, our aim was to determine whether a hepatic TG content lower than normal, as observed in HSL-knockout mice, leads to increased hepatic insulin sensitivity. Therefore, hyperinsulinemic clamp experiments in combination with D-(3)H-glucose were used. Furthermore, hepatic insulin receptor and phosphorylated protein kinase B (PKB-P)/akt were analyzed by Western blotting. No significant differences where observed in insulin-mediated whole-body glucose uptake between HSL-knockout and control mice. Interestingly, hepatic insulin sensitivity of HSL-knockout mice was increased, because insulin caused a greater reduction in endogenous glucose production ( approximately 71% compared with approximately 31% in control mice; P < 0.05), despite decreased plasma adiponectin levels. PKB/akt phosphorylation and phosphatidylinositol-3-kinase activity was significantly higher in livers of HSL-knockout mice after insulin stimulation. In HSL-knockout mice, reduced hepatic TG stores result in an increased suppressive effect of insulin on hepatic glucose production, in line with an increased hepatic PKB-P/akt and phosphatidylinositol-3 kinase activity. Thus, hepatic insulin sensitivity is indeed increased after reducing hepatic TG stores below normal.

Abstract: CD36 (fatty acid translocase) is involved in high-affinity peripheral fatty acid uptake. Mice lacking CD36 exhibit increased plasma free fatty acid and triglyceride (TG) levels and decreased glucose levels. Studies in spontaneous hypertensive rats lacking functional CD36 link CD36 to the insulin-resistance syndrome. To clarify the relationship between CD36 and insulin sensitivity in more detail, we determined insulin-mediated whole-body and tissue-specific glucose uptake in CD36-deficient (CD36-/-) mice. Insulin-mediated whole-body and tissue-specific glucose uptake was measured by d-[3H]glucose and 2-deoxy-d-[1-3H]glucose during hyperinsulinemic clamp in CD36-/- and wild-type control littermates (CD36+/+) mice. Whole-body and muscle-specific insulin-mediated glucose uptake was significantly higher in CD36-/- compared with CD36+/+ mice. In contrast, insulin completely failed to suppress endogenous glucose production in CD36-/- mice compared with a 40% reduction in CD36+/+ mice. This insulin-resistant state of the liver was associated with increased hepatic TG content in CD36-/- mice compared with CD36+/+ mice (110.9 +/- 12.0 and 68.9 +/- 13.6 microg TG/mg protein, respectively). Moreover, hepatic activation of protein kinase B by insulin, measured by Western blot, was reduced by 54%. Our results show a dissociation between increased muscle and decreased liver insulin sensitivity in CD36-/- mice.

Abstract: Increasing evidence has implicated the membrane protein CD36 (or fatty acid translocase, FAT) to be involved in high affinity fatty acid uptake. CD36 is expressed in tissues active in fatty acid metabolism, like adipose tissue and skeletal and cardiac muscle, but also in intestine. CD36 is localized in the intestine mainly in the jejunal villi, where it is confined to enterocyte apical membrane. The aim was to determine the role of CD36 in intestinal lipid absorption. Lipid absorption was determined by administering 3H-labeled triolein and 14C-labeled palmitic acid as an olive oil bolus by intragastric gavage and determine appearance of 3H and 14C label in plasma, after blocking lipolysis by i.v. injections of Triton WR 1339. Surprisingly, no differences in plasma appearance of 3H-label or 14C-label were observed in CD36(-/-) mice compared to wild type controls. These results suggest that CD36 does not play a role in intestinal lipid absorption after an acute lipid load.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract:

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract:

Abstract: The extent to which the transport of glucose across the plasma membrane of the yeast Saccharomyces bayanus controls the glycolytic flux was determined. The magnitude of control was quantified by measuring the effect of small changes in the activity of the glucose transport system on the rate of glucose consumption. Two effectors were used to modulate the activity of glucose transport: (i) maltose, a competitive inhibitor of the glucose transport system in S. bayanus (as well as in Saccharomyces cerevisiae) and (ii) extracellular glucose, the substrate of the glucose transport system. Two approaches were followed to derive from the experimental data the flux control coefficient of glucose transport on the glycolytic flux: (i) direct comparison of the steady-state glycolytic flux with the zero trans-influx of glucose and (ii) comparison of the change in glycolytic flux with the concomitant change in calculated glucose transport activity on variation of the extracellular glucose concentration. Both these approaches demonstrated that in cells of S. bayanus grown on glucose and harvested at the point of glucose exhaustion, a high proportion of the control of the glycolytic flux resides in the transport of glucose across the plasma membrane.

Abstract: 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.

Abstract: 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).

Abstract: In a population of intact cells of the yeast Saccharomyces cerevisiae the dynamics of glycolytic metabolism were investigated under the condition of sustained oscillations. At 5-s intervals cells were quenched in -40 degrees C methanol, extracted and the intracellular concentrations of glycolytic metabolites, adenine nucleotides and phosphate were analysed. Oscillations were found for the glycolytic intermediates glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate. At variance with earlier reports on transient glycolytic oscillations, some intermediates further down the glycolytic pathway did not oscillate significantly, even though NADH did. In addition, the adenylate energy charge and the free energy of ATP hydrolysis oscillated significantly. Dynamic coupling through the latter may be responsible for this effective compartmentation of glycolytic dynamics.

Abstract: In the presence of cyanide, populations of yeast cells can exhibit sustained oscillations in the concentration of glycolytic metabolites, NADH and ATP. This study attempts to answer the long-standing question of whether and how oscillations of individual cells are synchronized. It shows that mixing two cell populations that oscillate 180 degrees out of phase only transiently abolishes the macroscopic oscillation. After a few minutes, NADH fluorescence of the mixed population resumes oscillations up to the original amplitude. At low cell densities, addition of acetaldehyde causes transient oscillations. At higher cell densities, where the oscillations are autonomous, 70 microM acetaldehyde causes phase shifts. Extracellular acetaldehyde is shown to oscillate around the 70 microM level. We conclude that acetaldehyde synchronizes the oscillations of the individual cells.

Abstract: The three main existing models for glycolytic oscillations in yeast were re-examined to investigate how these oscillations are controlled. We implemented the operational definitions provided by metabolic control analysis to quantify the control properties of enzymes with regard to glycolytic oscillations. In all three models, the control of the frequency and that of the amplitudes of the metabolites were distributed among the enzymes. There was no obvious correlation between the control of the average flax and the control of the frequency. Most importantly, the so-called 'oscillophore' of the system, traditionally the enzyme primarily held responsible for the generation of the oscillation, was not the only controlling step. We conclude that just like steady-state flux control is not necessarily limited to a rate-limiting step, oscillations are not dictated by a single 'oscillophore'.

Abstract: 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.

Abstract: 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.

Abstract: Glycolytic oscillations can be induced by adding glucose to starved Saccharomyces cerevisiae cells and, after a steady state has been established, cyanide. Transient oscillations or limit-cycle oscillations can be induced depending on the growth phase in which the cells are harvested. To find what causes these differences in the dynamic behaviour, we analyzed glycolytic enzyme activities at different growth phases. The hexokinase activity increased by a factor of three after growth substrate transition from glucose to ethanol; the other measured activities remained constant. Cyanide was found not only to block respiration, but also to trap acetaldehyde. Both cyanide actions appear necessary for the occurrence of sustained glycolytic oscillations.

Abstract: The control of enzymes and substrates on the flux through microbial metabolic pathways can be quantified in terms of flux control coefficients. In pathways involving group transfer, the summation theorem for flux control by the enzymes has to be modified: the sum of control by all enzymes is between 1 and 2. The phosphoenolpyruvate:glucose phosphotransferase system is such a pathway. Experimental determination of the control by the enzymes in this pathway is under way. The control of the enzymes on the glycolytic flux in yeast is low, with the possible exception of the uptake step. In Klebsiella pneumoniae potassium and ammonium ions can simultaneously be 'limiting', (i.e. have significant control on growth) at pH6, but not at pH8. This may be due to the fact that at pH8 the high-affinity potassium uptake system is absent.

Abstract: Under a limited set of hitherto incompletely defined conditions, inhibition of respiration has been shown to cause transient oscillations in NAD(P)H fluorescence of yeast cells. In this paper, we apply a new method [1992, Anal. Biochem. 204, 118-132] for extraction of intracellular metabolites. This method involves spraying the cells into -40 degrees C methanol; the neutral pH allows extraction of nearly all intracellular metabolites, including NADH. Close to the shift from glucose to ethanol as a growth substrate, the cells acquire a make-up amenable to sustained oscillations in intracellular concentrations of NADH and glycolytic intermediates such as glucose-6-phosphate. NADH was found to oscillate between 200 microM and 400 microM intracellular concentration. The cellular make-up determining the tendency to oscillate is 'remembered' by the cells after three hours of starvation.