Metabolic Steady State Research Articles

Applying theories of microbial metabolism for induction of targeted enzyme activity in a methanogenic microbial community at a metabolic steady state.

Novel enzymes that are stable in diverse conditions are intensively sought because they offer major potential advantages in industrial biotechnology, and microorganisms in extreme environments are key sources of such enzymes. However, most potentially valuable enzymes are currently inaccessible due to the pure culturing problem of microorganisms. Novel metagenomic and metaproteomic techniques that circumvent the need for pure cultures have theoretically provided possibilities to identify all genes and all proteins in microbial communities, but these techniques have not been widely used to directly identify specific enzymes because they generate vast amounts of extraneous data.In a first step towards developing a metaproteomic approach to pinpoint targeted extracellular hydrolytic enzymes of choice in microbial communities, we have generated and analyzed the necessary conditions for such an approach by the use of a methanogenic microbial community maintained on a chemically defined medium. The results show that a metabolic steady state of the microbial community could be reached, at which the expression of the targeted hydrolytic enzymes were suppressed, and that upon enzyme induction a distinct increase in the targeted enzyme expression was obtained. Furthermore, no cross talk in expression was detected between the two focal types of enzyme activities under their respective inductive conditions. Thus, the described approach should be useful to generate ideal samples, collected before and after selective induction, in controlled microbial communities to clearly discriminate between constituently expressed proteins and extracellular hydrolytic enzymes that are specifically induced, thereby reducing the analysis to only those proteins that are distinctively up-regulated.

Approximating the stabilization of cellular metabolism by compartmentalization.

Biochemical regulation in compartmentalized metabolic networks is highly complex and non-intuitive. This is particularly true for cells of higher plants showing one of the most compartmentalized cellular structures across all kingdoms of life. The interpretation and testable hypothesis generation from experimental data on such complex systems is a challenging step in biological research and biotechnological applications. While it is known that subcellular compartments provide defined reaction spaces within a cell allowing for the tight coordination of complex biochemical reaction sequences, its role in the coordination of metabolic signals during metabolic reprogramming due to environmental fluctuations is less clear. In the present study, we numerically analysed the effects of environmental fluctuations in a subcellular metabolic network with regard to the stability of an experimentally observed steady state in the genetic model plant Arabidopsis thaliana. Applying a method for kinetic parameter normalization, several millions of probable enzyme kinetic parameter constellations were simulated and evaluated with regard to the stability information of the metabolic homeostasis. Information about the stability of the metabolic steady state was derived from real parts of eigenvalues of Jacobian matrices. Our results provide evidence for a differential stabilizing contribution of different subcellular compartments. We could identify stabilizing and destabilizing network components which we could classify according to their subcellular localization. The findings prove that a highly dynamic interplay between intracellular compartments is preliminary for an efficient stabilization of a metabolic homeostasis after environmental perturbation. Further, our results provide evidence that feedback-inhibition originating from the cytosol and plastid seem to stabilize the sucrose homeostasis more efficiently than vacuolar control. In summary, our results indicate stabilizing and destabilizing network components in context of their subcellular organization.Electronic supplementary materialThe online version of this article (doi:10.1007/s12064-016-0225-y) contains supplementary material, which is available to authorized users.

The relationship between retinol-binding protein 4 and apolipoprotein B-containing lipoproteins is attenuated in patients with very high serum triglycerides: A pilot study.

The investigation of the association between retinol-binding protein 4 (RBP4) and lipoproteins in subjects with hypertriglyceridemia. Forty-six obese or overweight hypertriglyceridemic patients were studied at baseline and 20 of them underwent a hypocaloric low-fat diet for 3 months. Plasma RBP4 levels were positively correlated with serum triglycerides (TG) in the subgroup of patients with TG <200 mg/dL (r=0.453, p=0.039) and negatively correlated with TG in patients with TG ≥200 mg/dL (r=-0.487, p=0.019). In the subgroup with TG <200 mg/ dL, subjects with circulating RBP4 above the median 46 mg/L had higher levels of intermediate density lipoprotein-cholesterol (IDL-C), low-density lipoprotein-cholesterol (LDL-C) and apolipoprotein B (ApoB), while these differences were absent in patients with TG ≥200 mg/dL. The associations of percentage changes of circulating RBP4 with the percentage changes of LDL-C, very low-density lipoprotein-cholesterol (VLDL-C) and ApoB were positive after the first month and 3 months of diet for patients with baseline TG <200 mg/dL, while no correlations existed for patients with TG ≥200 mg/dL. The positive association between circulating RBP4 and ApoB-containing lipoproteins in a steady metabolic state, as well as during a hypocaloric diet, appears to be attenuated in patients with very high TG.

Immortalized Parkinson's disease lymphocytes have enhanced mitochondrial respiratory activity.

ABSTRACTIn combination with studies of post-mortem Parkinson's disease (PD) brains, pharmacological and genetic models of PD have suggested that two fundamental interacting cellular processes are impaired – proteostasis and mitochondrial respiration. We have re-examined the role of mitochondrial dysfunction in lymphoblasts isolated from individuals with idiopathic PD and an age-matched control group. As previously reported for various PD cell types, the production of reactive oxygen species (ROS) by PD lymphoblasts was significantly elevated. However, this was not due to an impairment of mitochondrial respiration, as is often assumed. Instead, basal mitochondrial respiration and ATP synthesis are dramatically elevated in PD lymphoblasts. The mitochondrial mass, genome copy number and membrane potential were unaltered, but the expression of indicative respiratory complex proteins was also elevated. This explains the increased oxygen consumption rates by each of the respiratory complexes in experimentally uncoupled mitochondria of iPD cells. However, it was not attributable to increased activity of the stress- and energy-sensing protein kinase AMPK, a regulator of mitochondrial biogenesis and activity. The respiratory differences between iPD and control cells were sufficiently dramatic as to provide a potentially sensitive and reliable biomarker of the disease state, unaffected by disease duration (time since diagnosis) or clinical severity. Lymphoblasts from control and PD individuals thus occupy two distinct, quasi-stable steady states; a ‘normal’ and a ‘hyperactive’ state characterized by two different metabolic rates. The apparent stability of the ‘hyperactive’ state in patient-derived lymphoblasts in the face of patient ageing, ongoing disease and mounting disease severity suggests an early, permanent switch to an alternative metabolic steady state. With its associated, elevated ROS production, the ‘hyperactive’ state might not cause pathology to cells that are rapidly turned over, but brain cells might accumulate long-term damage leading ultimately to neurodegeneration and the loss of mitochondrial function observed post-mortem. Whether the ‘hyperactive’ state in lymphoblasts is a biomarker specifically of PD or more generally of neurodegenerative disease remains to be determined.

Probing Cancer Cell Metabolism Using NMR Spectroscopy.

Altered cellular metabolism is now accepted to be at the core of many diseases including cancer. Over the past 20 years, NMR has become a core technology to study these metabolic perturbations in detail. This chapter reviews current NMR-based methods for steady-state metabolism and, in particular, the use of non-radioactive stable isotope-enriched tracers. Opportunities and challenges for each method, such as 1D (1)H NMR spectroscopy and (13)C carbon-based NMR spectroscopic methods, are discussed. Ultimately, the combination of NMR and mass spectra as orthogonal technologies are required to compensate for the drawbacks of each technique when used singly are discussed.

Identification of active elementary flux modes in mitochondria using selectively permeabilized CHO cells

Metabolic compartmentation is a key feature of mammalian cells. Mitochondria are the powerhouse of eukaryotic cells, responsible for respiration and the TCA cycle. We accessed the mitochondrial metabolism of the economically important Chinese hamster ovary (CHO) cells using selective permeabilization. We tested key substrates without and with addition of ADP. Based on quantified uptake and production rates, we could determine the contribution of different elementary flux modes to the metabolism of a substrate or substrate combination. ADP stimulated the uptake of most metabolites, directly by serving as substrate for the respiratory chain, thus removing the inhibitory effect of NADH, or as allosteric effector. Addition of ADP favored substrate metabolization to CO2 and did not enhance the production of other metabolites. The controlling effect of ADP was more pronounced when we supplied metabolites to the first part of the TCA cycle: pyruvate, citrate, α-ketoglutarate and glutamine. In the second part of the TCA cycle, the rates were primarily controlled by the concentrations of C4-dicarboxylates. Without ADP addition, the activity of the pyruvate carboxylase–malate dehydrogenase–malic enzyme cycle consumed the ATP produced by oxidative phosphorylation, preventing its accumulation and maintaining metabolic steady state conditions. Aspartate was taken up only in combination with pyruvate, whose uptake also increased, a fact explained by complex regulatory effects. Isocitrate dehydrogenase and α-ketoglutarate dehydrogenase were identified as the key regulators of the TCA cycle, confirming existent knowledge from other cells. We have shown that selectively permeabilized cells combined with elementary mode analysis allow in-depth studying of the mitochondrial metabolism and regulation.

Advanced continuous cultivation methods for systems microbiology.

Increasing the throughput of systems biology-based experimental characterization of in silico-designed strains has great potential for accelerating the development of cell factories. For this, analysis of metabolism in the steady state is essential as only this enables the unequivocal definition of the physiological state of cells, which is needed for the complete description and in silico reconstruction of their phenotypes. In this review, we show that for a systems microbiology approach, high-resolution characterization of metabolism in the steady state--growth space analysis (GSA)--can be achieved by using advanced continuous cultivation methods termed changestats. In changestats, an environmental parameter is continuously changed at a constant rate within one experiment whilst maintaining cells in the physiological steady state similar to chemostats. This increases the resolution and throughput of GSA compared with chemostats, and, moreover, enables following of the dynamics of metabolism and detection of metabolic switch-points and optimal growth conditions. We also describe the concept, challenge and necessary criteria of the systematic analysis of steady-state metabolism. Finally, we propose that such systematic characterization of the steady-state growth space of cells using changestats has value not only for fundamental studies of metabolism, but also for systems biology-based metabolic engineering of cell factories.

Refined elasticity sampling for Monte Carlo-based identification of stabilizing network patterns.

Motivation: Structural kinetic modelling (SKM) is a framework to analyse whether a metabolic steady state remains stable under perturbation, without requiring detailed knowledge about individual rate equations. It provides a representation of the system’s Jacobian matrix that depends solely on the network structure, steady state measurements, and the elasticities at the steady state. For a measured steady state, stability criteria can be derived by generating a large number of SKMs with randomly sampled elasticities and evaluating the resulting Jacobian matrices. The elasticity space can be analysed statistically in order to detect network positions that contribute significantly to the perturbation response. Here, we extend this approach by examining the kinetic feasibility of the elasticity combinations created during Monte Carlo sampling.Results: Using a set of small example systems, we show that the majority of sampled SKMs would yield negative kinetic parameters if they were translated back into kinetic models. To overcome this problem, a simple criterion is formulated that mitigates such infeasible models. After evaluating the small example pathways, the methodology was used to study two steady states of the neuronal TCA cycle and the intrinsic mechanisms responsible for their stability or instability. The findings of the statistical elasticity analysis confirm that several elasticities are jointly coordinated to control stability and that the main source for potential instabilities are mutations in the enzyme alpha-ketoglutarate dehydrogenase.Contact: dorothee.childs@embl.deSupplementary information: Supplementary data are available at Bioinformatics online.

Suppressing the Neurospora crassa circadian clock while maintaining light responsiveness in continuous stirred tank reactors.

Neurospora crassa has been utilized as a model organism for studying biological, regulatory, and circadian rhythms for over 50 years. These circadian cycles are driven at the molecular level by gene transcription events to prepare for environmental changes. N. crassa is typically found on woody biomass and is commonly studied on agar-containing medium which mimics its natural environment. We report a novel method for disrupting circadian gene transcription while maintaining light responsiveness in N. crassa when held in a steady metabolic state using bioreactors. The arrhythmic transcription of core circadian genes and downstream clock-controlled genes was observed in constant darkness (DD) as determined by reverse transcription-quantitative PCR (RT-qPCR). Nearly all core circadian clock genes were up-regulated upon exposure to light during 11hr light/dark cycle experiments under identical conditions. Our results demonstrate that the natural timing of the robust circadian clock in N. crassa can be disrupted in the dark when maintained in a consistent metabolic state. Thus, these data lead to a path for the production of industrial scale enzymes in the model system, N. crassa, by removing the endogenous negative feedback regulation by the circadian oscillator.

Steady-state metabolism of ethanol in perfused rat livers treated with cyanamide: quantitative analysis of acetaldehyde effects on the metabolic flux rates.

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are principal enzymes responsible for metabolism of ethanol in mammals. The steady-state metabolic flux of ethanol has been poorly understood. We investigated flux rates of the individual steps of ethanol metabolism in perfused rat livers treated with ALDH inactivator cyanamide as an attempt to mimic human ALDH2 deficiency commonly seen in East Asians. The net rates of ethanol oxidation, acetaldehyde oxidation, and acetate activation were determined with a set of defined equations, based on the set influx rates of ethanol and the measured efflux rates of ethanol, acetaldehyde, and acetate. After intraperitoneal injections of 0.2 and 1.5mg/kg cyanamide, hepatic activities of mitochondrial ALDH2 and cytoplasmic ALDH1A1 decreased to a similar degree, that is, 51 to 57% and 69 to 74%, compared with the corresponding controls, respectively, whereas cytoplasmic ADH1 activity remained unchanged. At infusing 2mM ethanol, acetaldehyde oxidation rate well matched (99%) the net ethanol oxidation rate in control liver. Both the ethanol and acetaldehyde oxidation rates were significantly decreased after cyanamide treatments. At 10mM ethanol, the efflux acetaldehyde was significantly higher than that infusing 2mM ethanol in both control and cyanamide groups. Seventy-eight percent of the oxidized ethanol released as efflux acetate. At 2mM ethanol, the apparent flux control coefficients of ADH1 were assessed to be 0.78, 0.54, and 0.39, respectively, in control, low, and high cyanamide-treated livers. Kinetic simulations revealed that inhibition by acetaldehyde may largely account for the observed reduction of ADH1 oxidation rates after cyanamide treatment. Our results provide the first flux evidence that ADH and ALDH are steps influencing steady-state metabolism of ethanol in rat livers with inactivated ALDHs.

Fast sampling method for mammalian cell metabolic analyses using liquid chromatography-mass spectrometry.

Metabolomics has emerged as a powerful tool for addressing biological questions. Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used for metabolic characterization, including targeted and untargeted approaches. Despite recent innovations, a crucial aspect of this technique is the sample preparation for accurate data analyses. In this protocol, we present a robust and adaptable workflow for metabolic analyses of mammalian cells from adherent cell cultures, which is particularly suited for qualitative and quantitative central metabolite characterization by LC-MS. Each sample consists of 600,000 mammalian cells grown on cover glasses, allowing for fast and complete transfer of the cells for metabolite extraction or medium exchange, e.g., for labeling experiments. The sampling procedure includes a fast and efficient washing step in liquid flow in water, which reduces cross-contamination and matrix effects while minimizing perturbation of the metabolic steady state of the cells; it is followed by quenching cell metabolism. The latter is achieved by using a -20 °C cold methanol acetonitrile mixture acidified with formic acid, followed by freeze drying, metabolite extraction and LC-MS. The protocol requires 2 s for cell sampling until quenching, and the entire protocol takes a total of 1.5 h per sample when the provided nanoscale LC-MS method is applied.

La acción antifatigante de la cocaina y la habituación a la coca en el Perú

Se establece primero en amplios términos, el concepto de fatiga, recalcando que se trata de un proceso susceptible de ser desglosado en una serie de eslabones, siendo los principales : la fatiga de las sinápsis centrales, la fatiga de la placa neuro-muscular y la fatiga de la fibra muscular. La fatiga de la sinápsis centrales relacionadas con el movimiento, tiene su explicación en la ruptura de algunos de los estados de equilibrio químico que caracterizan a la función de la neurona. Está localizada en la sinápsis y su esencia es la incapacidad para trasmitir los impulsos nerviosos a través de los diversos eslabones neuronales. En esquema, se trata de una falla de los intermediarios químicos para adquirir la concentración necesaria para trasmitir el impulso, o de una incapacidad de la neurona para ser excitada por una concentración normal de intermediario. La disminución del umbral de las neuronas, producirá en teoría, la disminución de la fatiga. La fatiga mental, se comporta, aparentemente, suigiendo las mismas reglas generales. En la placa neuromuscular, los trabajos prolongados suponen también el mantenimiento de una serie de estados de equilibrio metabólico, siendo el principal el metabolismo acetilcolínico. Juegan también un importante rol los iones. La fatiga de la fibra muscular, presupone la existencia de grandes desequilibrios metabólicos que nunca se presentan en el organismo normal, por impedirlo la fatiga del Sistema Nervioso Central y de la placa neuro-muscular. Los procesos metabólicos relacionados con la contracción muscular, juegan, sin embargo, un rol primordial en el desarrollo de la fatoga total, producida por una incapacidad de los sistemas de recuperación para pagar las deudas metabólicas contraidas por los músculos. Los diversos eslabones de la fatiga, se encuentran íntimamente relacionadas en el organismo normal, dependiendo mutuamente uno de otro.

Transcriptomic and biochemical evidence for the role of lysine biosynthesis against linoleic acid hydroperoxide-induced stress in Saccharomyces cerevisiae

Amino acid biosynthesis forms part of an integrated stress response against oxidants in Saccharomyces cerevisiae and higher eukaryotes. Here we show an essential protective role of the l-lysine biosynthesis pathway in response to the oxidative stress condition induced by the lipid oxidant-linoleic acid hydroperoxide (LoaOOH), by means of transcriptomic profiling and phenotypic analysis, and using the deletion mutant dal80∆ and lysine auxotroph lys1∆. A comprehensive up-regulation of lysine biosynthetic genes (LYS1, LYS2, LYS4, LYS9, LYS12, LYS20 and LYS21) was revealed in dal80Δ following the oxidant challenge. The lysine auxotroph (lys1∆) exhibited a significant decrease in growth compared with that of BY4743 upon exposure to LoaOOH, albeit with the sufficient provision of lysine in the medium. Furthermore, the growth of wild type BY4743 exposed to LoaOOH was also greatly reduced in lysine-deficient conditions, despite a full complement of lysine biosynthetic genes. Amino acid analysis of LoaOOH-treated yeast showed that the level of cellular lysine remained unchanged throughout oxidant challenge, suggesting that the induced lysine biosynthesis leads to a steady-state metabolism as compared to the untreated yeast cells. Together, these findings demonstrate that lysine availability and its biosynthesis pathway play an important role in protecting the cell from lipid peroxide-induced oxidative stress, which is directly related to understanding environmental stress and industrial yeast management in brewing, wine making and baking.

A coupled thermodynamic and metabolic control analysis methodology and its evaluation on glycerol biosynthesis in Saccharomyces cerevisiae.

A coupled in silico thermodynamic and probabilistic metabolic control analysis methodology was verified by applying it to the glycerol biosynthetic pathway in Saccharomyces cerevisiae. The methodology allows predictions even when detailed knowledge of the enzyme kinetics is lacking. In a metabolic steady state, we found that glycerol-3-phosphate dehydrogenase operates far from thermodynamic equilibrium ([Formula: see text] -15.9 to -47.5 kJ mol(-1), where [Formula: see text] is the transformed Gibbs energy of the reaction). Glycerol-3-phosphataseoperates in modes near the thermodynamic equilibrium, far from the thermodynamic equilibrium or in between ([Formula: see text] ≈ 0 to -23.7 kJ mol(-1)). From the calculated distribution of the scaled flux control coefficients (median = 0.81), we inferred that the pathway flux is primarily controlled by glycerol-3-phosphate dehydrogenase. This prediction is consistent with previous findings, verifying the efficacy of the proposed methodology.

Modulation of olfactory sensitivity and glucose-sensing by the feeding state in obese Zucker rats.

The Zucker fa/fa rat has been widely used as an animal model to study obesity, since it recapitulates most of its behavioral and metabolic dysfunctions, such as hyperphagia, hyperglycemia and insulin resistance. Although it is well established that olfaction is under nutritional and hormonal influences, little is known about the impact of metabolic dysfunctions on olfactory performances and glucose-sensing in the olfactory system of the obese Zucker rat. In the present study, using a behavioral paradigm based on a conditioned olfactory aversion, we have shown that both obese and lean Zucker rats have a better olfactory sensitivity when they are fasted than when they are satiated. Interestingly, the obese Zucker rats displayed a higher olfactory sensitivity than their lean controls. By investigating the molecular mechanisms involved in glucose-sensing in the olfactory system, we demonstrated that sodium-coupled glucose transporters 1 (SGLT1) and insulin dependent glucose transporters 4 (GLUT4) are both expressed in the olfactory bulb (OB). By comparing the expression of GLUT4 and SGLT1 in OB of obese and lean Zucker rats, we found that only SGLT1 is regulated in genotype-dependent manner. Next, we used glucose oxidase biosensors to simultaneously measure in vivo the extracellular fluid glucose concentrations ([Gluc]ECF) in the OB and the cortex. Under metabolic steady state, we have determined that the OB contained twice the amount of glucose found in the cortex. In both regions, the [Gluc]ECF was 2 fold higher in obese rats compared to their lean controls. Under induced dynamic glycemia conditions, insulin injection produced a greater decrease of [Gluc]ECF in the OB than in the cortex. Glucose injection did not affect OB [Gluc]ECF in Zucker fa/fa rats. In conclusion, these results emphasize the importance of glucose for the OB network function and provide strong arguments towards establishing the OB glucose-sensing as a key factor for sensory olfactory processing.

A mathematical model of metabolism and regulation provides a systems-level view of how Escherichia coli responds to oxygen.

The efficient redesign of bacteria for biotechnological purposes, such as biofuel production, waste disposal or specific biocatalytic functions, requires a quantitative systems-level understanding of energy supply, carbon, and redox metabolism. The measurement of transcript levels, metabolite concentrations and metabolic fluxes per se gives an incomplete picture. An appreciation of the interdependencies between the different measurement values is essential for systems-level understanding. Mathematical modeling has the potential to provide a coherent and quantitative description of the interplay between gene expression, metabolite concentrations, and metabolic fluxes. Escherichia coli undergoes major adaptations in central metabolism when the availability of oxygen changes. Thus, an integrated description of the oxygen response provides a benchmark of our understanding of carbon, energy, and redox metabolism. We present the first comprehensive model of the central metabolism of E. coli that describes steady-state metabolism at different levels of oxygen availability. Variables of the model are metabolite concentrations, gene expression levels, transcription factor activities, metabolic fluxes, and biomass concentration. We analyze the model with respect to the production capabilities of central metabolism of E. coli. In particular, we predict how precursor and biomass concentration are affected by product formation.

A Test of the Metabolic Cost of Cushioning Hypothesis during Unshod and Shod Running

This study aimed to investigate the effects of surface and shoe cushioning on the metabolic cost of running. In running, the leg muscles generate force to cushion the impact with the ground. External cushioning (surfaces or shoes) may reduce the muscular effort needed for cushioning and thus reduce metabolic cost. Our primary hypothesis was that the metabolic cost of unshod running would decrease with a more cushioned running surface. We also hypothesized that because of the counteracting effects of shoe cushioning and mass, unshod running on a hard surface would have approximately the same metabolic cost as running in lightweight, cushioned shoes. To test these hypotheses, we attached 10- and 20-mm-thick slats of the same foam cushioning used in running shoe midsoles to the belt of a treadmill that had a rigid deck. Twelve subjects who preferred a midfoot strike pattern and had substantial barefoot/minimalist running experience ran without shoes on the normal treadmill belt and on each thickness of foam. They also ran with lightweight, cushioned shoes on the normal belt. We collected V˙O2 and V˙CO2 to calculate the metabolic power demand and used a repeated-measures ANOVA to compare between conditions. Compared to running unshod on the normal belt, running unshod on the 10-mm-thick foam required 1.63% ± 0.67% (mean ± SD) less metabolic power (P = 0.034) but running on the 20-mm-thick foam had no significant metabolic effect. Running with and without shoes on the normal belt had similar metabolic power demands, likely because the beneficial energetic effects of cushioning counterbalanced the detrimental effects of shoe mass. On average, surface and shoe cushioning reduce the metabolic power required for submaximal running.

The Metabolic Modulator Perhexiline Induces Calcium-Cycling Dysfunction and Apoptosis in Cardiomyocyte Syncytia

Major pathogenic adaptations in chronic heart failure (CHF) include the reversion to glycolytic metabolism and the persistence of an energy deficient state. The use of metabolic modulators to increase the efficiency of glycolytic ATP production has emerged as a new therapeutic approach in CHF. Perhexiline (PHX), an anti-anginal agent, improves myocardial function in CHF patients by a mechanism that is thought to involve the inhibition of carnitine palmitoyl transferase (CPT) which prevents mitochondrial free fatty acid uptake. In silico modelling and cell-based experimental data predict that such alterations to the metabolic steady state would drive other compensatory (mal)adaptations that could negatively impact on cellular phenotype. Here, we investigated the phenotypic consequences of PHX-mediated metabolic modulation on Ca2+ cycling and apoptotic susceptibility in HL-1 syncytia. HL-1 cardiomyocytes exhibit a metabolic profile similar to that observed in CHF myocardium (glycolysis >> fatty acid oxidation). Contrary to the expected enhancement of Ca2+ cycling, PHX profoundly deranged Ca2+ signalling in a time- and concentration-dependent manner. The spatio-temporal organization and intercellular synchronization of Ca2+ oscillations was progressively reduced at [PHX] between 0.1 and 2.5μM and contractility was abolished at [PHX] > 5μM. These concentrations are below the levels of drug reported to accumulate in heart tissue following therapeutic dosing. Metabolic activity was compromised in PHX-treated cells and we determined PHX concentration-dependent caspase-linked apoptosis (EC50 ≈ 25μM). Oxfenicine (a potent CPT1 inhibitor) did not recapitulate any of these effects at concentrations up to 100μM. Our data show that, in the context of glycolytic metabolism, PHX provoked dysfunctional Ca2+ handling and increased cardiomyocyte susceptibility to apoptosis. These effects appeared to be independent of CPT inhibition.

Incorporation of flexible objectives and time-linked simulation with flux balance analysis

We present two modifications of the flux balance analysis (FBA) metabolic modeling framework which relax implicit assumptions of the biomass reaction. Our flexible flux balance analysis (flexFBA) objective removes the fixed proportion between reactants, and can therefore produce a subset of biomass reactants. Our time-linked flux balance analysis (tFBA) simulation removes the fixed proportion between reactants and byproducts, and can therefore describe transitions between metabolic steady states. Used together, flexFBA and tFBA model a time scale shorter than the regulatory and growth steady state encoded by the biomass reaction. This combined short-time FBA method is intended for integrated modeling applications to enable detailed and dynamic depictions of microbial physiology such as whole-cell modeling. For example, when modeling Escherichia coli, it avoids artifacts caused by low-copy-number enzymes in single-cell models with kinetic bounds. Even outside integrated modeling contexts, the detailed predictions of flexFBA and tFBA complement existing FBA techniques. We show detailed metabolite production of in silico knockouts used to identify when correct essentiality predictions are made for the wrong reason.

Salt sensitivity of renin secretion, glomerular filtration rate and blood pressure in conscious Sprague‐Dawley rats

We hypothesized that in normal rats in metabolic steady state, (i) the plasma renin concentration (PRC) is log-linearly related to Na(+) intake (NaI), (ii) the concurrent changes in mean arterial pressure (MABP) and glomerular filtration rate (GFR) are negligible and (iii) the function PRC = f(NaI) is altered by β₁-adrenoceptor blockade (metoprolol) and surgical renal denervation (DNX). In catheterized, conscious rats on low-Na(+) diet (0.004% Na(+)), NaI was increased by up to 120-fold, in four 3-day steps, by intravenous saline infusion. MABP was recorded continuously, PRC measured in arterial blood, and GFR estimated by inulin clearance. Steady states were achieved within 3 days. PRC [mIU L(-1)] was log-linearly related to NaI [mmol kg(-1) day(-1)]: PRC = -9.9 log (NaI) + 22. Set point (22 mIU L(-1) at NaI = 1) and slope (9.9 mIU per decade NaI) were independent of metoprolol administration and DNX. MABP and GFR were markedly salt-sensitive: MABP [mmHg] = 4.9 log (NaI) + 99 (P < 0.01), and GFR [mL min(-1)] = 1.4 log (NaI) + 8.3 (P < 0.01). MABP increased similarly (approx. 10%, P < 0.001) irrespective of pre-treatment. Metoprolol, but not DNX, reduced MABP, HR, and GFR (all P < 0.01). Salt sensitivity of GFR was not observed in DNX rats. Log-linear relations to sodium intake exist not only for PRC, but also for MABP and GFR, which per 10-fold increase in sodium intake rose by 5 mmHg and 1.4 mL min(-1) respectively. Steady-state levels of PRC appear independent of renal nerves. MABP and GFR seem markedly salt sensitive in normal rats.