Metabolic adaptation to the chronic loss of Ca2+ signaling induced by KO of IP3 receptors or the mitochondrial Ca2+ uniporter

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP₃Rs) and the mitochondrial Ca²⁺ uniporter (MCU) are key proteins that regulate intracellular Ca²⁺ concentration. Mitochondrial Ca²⁺ accumulation activates Ca²⁺-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP₃R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca²⁺ signaling. Our results show that TKO cells (exhibiting total loss of Ca²⁺ signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca²⁺ signaling is dispensable for the bioenergetic needs of both IP₃R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.     

Viral effects on metabolism: changes in glucose and glutamine utilization during human cytomegalovirus infection

Human cytomegalovirus (HCMV) infection causes dramatic alterations of intermediary metabolism, similar to those found in tumor cells. In infected cells, glucose carbon is not completely broken down by the tricarboxylic acid (TCA) cycle for energy; instead, it is used biosynthetically. This process requires increased glucose uptake, increased glycolysis and the diversion of glucose carbon, in the form of citrate, from the TCA cycle for use in HCMV-induced fatty acid biosynthesis. The diversion of citrate from the TCA cycle (cataplerosis) requires induction of enzymes to promote glutaminolysis, the conversion of glutamine to α-ketoglutarate to maintain the TCA cycle (anaplerosis) and ATP production. Such changes could result in heretofore uncharacterized pathogenesis, potentially implicating HCMV as a subtle cofactor in many maladies, including oncogenesis. Recognition of the effects of HCMV, and other viruses, on host cell metabolism will provide new understanding of viral pathogenesis and novel avenues for antiviral therapy.     

Pluripotent stem cell energy metabolism: an update

Recent studies link changes in energy metabolism with the fate of pluripotent stem cells (PSCs). Safe use of PSC derivatives in regenerative medicine requires an enhanced understanding and control of factors that optimize in vitro reprogramming and differentiation protocols. Relative shifts in metabolism from naïve through “primed” pluripotent states to lineage‐directed differentiation place variable demands on mitochondrial biogenesis and function for cell types with distinct energetic and biosynthetic requirements. In this context, mitochondrial respiration, network dynamics, TCA cycle function, and turnover all have the potential to influence reprogramming and differentiation outcomes. Shifts in cellular metabolism affect enzymes that control epigenetic configuration, which impacts chromatin reorganization and gene expression changes during reprogramming and differentiation. Induced PSCs (iPSCs) may have utility for modeling metabolic diseases caused by mutations in mitochondrial DNA, for which few disease models exist. Here, we explore key features of PSC energy metabolism research in mice and man and the impact this work is starting to have on our understanding of early development, disease modeling, and potential therapeutic applications.     

In Vivo and In Vitro Metabolomic Analysis of Anaerobic Rice Coleoptiles Revealed Unexpected Pathways

The ability of the rice (Oryza sativa L.) seedling to tolerate extended hypoxia during submergence is largely attributed to the biochemical adaptation of its coleoptile. Rice coleoptiles are capable of sustaining ATP production and cytoplasmic pH, unlike flood-sensitive organs, such as maize shoots. Fermentation reactions leading to the production of ethanol, alanine, succinate, and -aminobutyrate (GAB) are active in both types of tissues and thus may not account for the difference in tolerance. We have shown previously that rice coleoptiles undergo nitrate reduction and metabolism, which is efficient in alleviating cytoplasmic acidosis and regenerating NAD. Here, we employed ¹³C-2-acetate tracer methods with in vivo ¹³C NMR measurement, including in vivo isotopomer analysis, to probe the tricarboxylic acid (TCA) cycle and interacting pathways in rice coleoptiles during anaerobiosis. We found that the TCA cycle underwent multiple turns based on the metabolic scrambling of ¹³C label patterns in glutamine and malate. The in vivo kinetics of the ¹³C label incorporation into glutamic acid, glutamine, and GAB supports a separate pool of glutamate that was derived from the glutamate dehydrogenase reaction and subsequently decarboxylated to yield GAB. Both reactions consume additional H⁺ and/or NADH. Moreover, the higher rate of ¹³C enrichment at C-3 than C-2 of malate suggests the contribution of the glyoxylate cycle to malate synthesis, which could replenish the TCA cycle carbons diverted to GAB, glutamate, and glutamine synthesis. All of the above reactions contribute to the maintenance of glycolysis for energy production.     

The oxidative TCA cycle operates during methanotrophic growth of the Type I methanotroph Methylomicrobium buryatense 5GB1

Methanotrophs are a group of bacteria that use methane as sole carbon and energy source. Type I methanotrophs are gamma-proteobacterial methanotrophs using the ribulose monophosphate cycle (RuMP) cycle for methane assimilation. In order to facilitate metabolic engineering in the industrially promising Type I methanotroph Methylomicrobium buryatense 5GB1, flux analysis of cellular metabolism is needed and ¹³C tracer analysis is a foundational tool for such work. This biological system has a single-carbon input and a special network topology that together pose challenges to the current well-established methodology for ¹³C tracer analysis using a multi-carbon input such as glucose, and to date, no ¹³C tracer analysis of flux in a Type I methanotroph has been reported. In this study, we showed that by monitoring labeling patterns of several key intermediate metabolites in core metabolism, it is possible to quantitate the relative flux ratios for important branch points, such as the malate node. In addition, it is possible to assess the operation of the TCA cycle, which has been thought to be incomplete in Type I methanotrophs. Surprisingly, our analysis provides direct evidence of a complete, oxidative TCA cycle operating in M. buryatense 5GB1 using methane as sole carbon and energy substrate, contributing about 45% of the total flux for de novo malate production. Combined with mutant analysis, this method was able to identify fumA (METBUDRAFT_1453/MBURv2__60244) as the primary fumarase involved in the oxidative TCA cycle, among 2 predicted fumarases, supported by ¹³C tracer analysis on both fumA and fumC single knockouts. Interrupting the oxidative TCA cycle leads to a severe growth defect, suggesting that the oxidative TCA cycle functions to not only provide precursors for de novo biomass synthesis, but also to provide reducing power to the system. This information provides new opportunities for metabolic engineering of M. buryatense for the production of industrially relevant products.     

Terminal oxidations in Bacillus brevis ATCC 10068

Summary 1. Evidence is presented that contrary to previous reports a number of enzymes implicated in the TCA cycle are functional in an antibiotic producing strain of Bacillus brevis. 2. The level of enzyme activites recorded was found to be influenced by the permeability state of the cell membrane and by the presence of an NAD⁺/NADH degrading enzyme. These factors are discussed in relation to the previously reported negative findings of other workers. 3. The distribution and specific activities of TCA cycle enzymes in membrane and supernatant fractions are reported. 4. The results, by comparison with other organisms known to possess a functional TCA cycle, suggest that the cycle provides a major pathway of energy metabolism in B. brevis.     

Embryonic Lethality of Mitochondrial Pyruvate Carrier 1 Deficient Mouse Can Be Rescued by a Ketogenic Diet

Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic profiles, and both defects could be restored by reexpression of MPC1. Labeling experiments using ¹³C-labeled glucose and glutamine demonstrated that MPC deficiency causes increased glutaminolysis and reduced contribution of glucose-derived pyruvate to the TCA cycle. Morphological defects were observed in mutant embryonic brains, together with major alterations of their metabolome including lactic acidosis, diminished TCA cycle intermediates, energy deficit and a perturbed balance of neurotransmitters. Strikingly, these changes were reversed when the pregnant dams were fed a ketogenic diet, which provides acetyl-CoA directly to the TCA cycle and bypasses the need for a functional MPC. This allowed the normal gestation and development of MPC deficient pups, even though they all died within a few minutes post-delivery. This study establishes the MPC as a key player in regulating the metabolic state necessary for embryonic development, neurotransmitter balance and post-natal survival.     

Consistent Estimation of Gibbs Energy Using Component Contributions

Standard Gibbs energies of reactions are increasingly being used in metabolic modeling for applying thermodynamic constraints on reaction rates, metabolite concentrations and kinetic parameters. The increasing scope and diversity of metabolic models has led scientists to look for genome-scale solutions that can estimate the standard Gibbs energy of all the reactions in metabolism. Group contribution methods greatly increase coverage, albeit at the price of decreased precision. We present here a way to combine the estimations of group contribution with the more accurate reactant contributions by decomposing each reaction into two parts and applying one of the methods on each of them. This method gives priority to the reactant contributions over group contributions while guaranteeing that all estimations will be consistent, i.e. will not violate the first law of thermodynamics. We show that there is a significant increase in the accuracy of our estimations compared to standard group contribution. Specifically, our cross-validation results show an 80% reduction in the median absolute residual for reactions that can be derived by reactant contributions only. We provide the full framework and source code for deriving estimates of standard reaction Gibbs energy, as well as confidence intervals, and believe this will facilitate the wide use of thermodynamic data for a better understanding of metabolism.     

Mitochondrial aconitase is a key regulator of energy production for growth and protein expression in Chinese hamster ovary cells

Introduction

Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production.

Objectives

The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity.

Methods

To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi.

Results

Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production.

Conclusion

Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.

     

Coordination of energy and carbon metabolism in lysine producing Brevibacterium strains

The present paper deals with the coordination of energy metabolism, glucose consumption rate, glycolytic and TCA cycle enzyme activities in the lysine-producing bacterium Brevibacterium flavum. It is shown, that inhibition of the elctron transport chain causes changes of the following sequence:

at first, TCA cycle enzymes are activated;

secondly, TCA cycle enzyme activity decreases, and glycolytic enzyme activities as well as glucose transport rate increase; there is a slight increase in Q_o2 and a considerable one of O₂ consumption in cyanide-resistant respiration pathway;

thirdly, TCA cycle enzyme activities and glucose transport rate decrease.

It is supposed, that coordination of carbon and energy metabolism in B. flavum depends on intracellular ATP concentration or energy charge value.     

Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of Escherichia coli Central Carbon Metabolism

The response of Escherichia coli central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional (13)C-(1)H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of E. coli strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in E. coli cells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.     

Extramitochondrial tricarboxylic acid cycle in retinal rod outer segments

Vertebrate retinal rod Outer Segments (OS) are the site of visual transduction, an energy demanding process for which mechanisms of ATP supply are still poorly known. Glycolysis or diffusion of either ATP or phosphocreatine from the Inner Segment (IS) does not seem to display adequate timing to supply ATP for phototransduction. We have previously reported data suggesting an aerobic metabolism in OS, which would largely account for the light-stimulated ATP need of the photoreceptor.Here, by oxymetry and biochemical analyses we show that: (i) disks isolated by Ficoll flotation consume O₂ in the presence of physiological respiring substrates either in coupled or uncoupled conditions; (ii) OS homogenates contain the whole biochemical machinery for the degradation of glucose, i.e. glycolysis and the tricarboxylic acid cycle (TCA cycle), consistently with the results of our previous proteomic study. Activities of the 8 TCA cycle enzymes in OS were comparable to those in retinal mitochondria-enriched fractions. Disk and OS preparations were subjected to TEM analysis, and while they can be considered free of inner segment contaminants, immunogold with specific antibodies demonstrate the expression therein of both the visual pigment rhodopsin and F_oF₁-ATP synthase. Finally, double immunofluorescence on mouse retina sections demonstrated a colocalization of some respiratory complex mitochondrial proteins with rhodopsin in rod OS.Data, suggestive of the exportability of the mitochondrial machinery for aerobic metabolism, may shed light on those retinal pathologies related to energy supply impairment in OS and to mutations in TCA enzymes.     

Reconstruction and flux analysis of coupling between metabolic pathways of astrocytes and neurons: application to cerebral hypoxia

Background

It is a daunting task to identify all the metabolic pathways of brain energy metabolism and develop a dynamic simulation environment that will cover a time scale ranging from seconds to hours. To simplify this task and make it more practicable, we undertook stoichiometric modeling of brain energy metabolism with the major aim of including the main interacting pathways in and between astrocytes and neurons.

Model

The constructed model includes central metabolism (glycolysis, pentose phosphate pathway, TCA cycle), lipid metabolism, reactive oxygen species (ROS) detoxification, amino acid metabolism (synthesis and catabolism), the well-known glutamate-glutamine cycle, other coupling reactions between astrocytes and neurons, and neurotransmitter metabolism. This is, to our knowledge, the most comprehensive attempt at stoichiometric modeling of brain metabolism to date in terms of its coverage of a wide range of metabolic pathways. We then attempted to model the basal physiological behaviour and hypoxic behaviour of the brain cells where astrocytes and neurons are tightly coupled.

Results

The reconstructed stoichiometric reaction model included 217 reactions (184 internal, 33 exchange) and 216 metabolites (183 internal, 33 external) distributed in and between astrocytes and neurons. Flux balance analysis (FBA) techniques were applied to the reconstructed model to elucidate the underlying cellular principles of neuron-astrocyte coupling. Simulation of resting conditions under the constraints of maximization of glutamate/glutamine/GABA cycle fluxes between the two cell types with subsequent minimization of Euclidean norm of fluxes resulted in a flux distribution in accordance with literature-based findings. As a further validation of our model, the effect of oxygen deprivation (hypoxia) on fluxes was simulated using an FBA-derivative approach, known as minimization of metabolic adjustment (MOMA). The results show the power of the constructed model to simulate disease behaviour on the flux level, and its potential to analyze cellular metabolic behaviour in silico.

Conclusion

The predictive power of the constructed model for the key flux distributions, especially central carbon metabolism and glutamate-glutamine cycle fluxes, and its application to hypoxia is promising. The resultant acceptable predictions strengthen the power of such stoichiometric models in the analysis of mammalian cell metabolism.     

Derivatization of the tricarboxylic acid intermediates with O-benzylhydroxylamine for liquid chromatography–tandem mass spectrometry detection

The tricarboxylic acid (TCA) cycle is an interface among glycolysis, lipid metabolism, and amino acid metabolism. Increasing interest in cancer metabolism has created a demand for rapid and sensitive methods for quantifying the TCA cycle intermediates and related organic acids. We have developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to quantify the TCA cycle intermediates in a 96-well format after O-benzylhydroxylamine (O-BHA) derivatization under aqueous conditions. This method was validated for quantitation of all common TCA cycle intermediates with good sensitivity, including α-ketoglutarate, malate, fumarate, succinate, 2-hydroxyglutarate, citrate, oxaloacetate, pyruvate, isocitrate, and lactate using a 8-min run time in cancer cells and tissues. The method was used to detect and quantify changes in metabolite levels in cancer cells and tumor tissues treated with a pharmacological inhibitor of nicotinamide phosphoribosyl transferase (NAMPT). This method is rapid, sensitive, and reproducible, and it can be used to assess metabolic changes in cancer cells and tumor samples.     

The tricarboxylic acid cycle in L₃ Teladorsagia circumcincta: metabolism of acetyl CoA to succinyl CoA

Nematodes, like other species, derive much of the energy for cellular processes from mitochondrial pathways including the TCA cycle. Previously, we have shown L₃Teladorsagia circumcincta consume oxygen and so may utilise a full TCA cycle for aerobic energy metabolism. We have assessed the relative activity levels and substrate affinities of citrate synthase, aconitase, isocitrate dehydrogenase (both NAD⁺ and NADP⁺ specific) and α-ketoglutarate dehydrogenase in homogenates of L₃T. circumcincta. All of these enzymes were present in homogenates. Compared with citrate synthase, low levels of enzyme activity and low catalytic efficiency was observed for NAD⁺ isocitrate dehydrogenase and especially α-ketoglutarate dehydrogenase. Therefore, it is likely that the activity of these to enzymes regulate overall metabolite flow through the TCA cycle, especially when [NAD⁺] limits enzyme activity. Of the enzymes tested, only citrate synthase had substrate affinities which were markedly different from values obtained from mammalian species. Overall, the results are consistent with the suggestion that a full TCA cycle exists within L₃T. circumcincta. While there may subtle variations in enzyme properties, particularly for citrate synthase, the control points for the TCA cycle in L₃T. circumcincta are probably similar to those in the tissues of their host species.     

Reconstitution of oxaloacetate metabolism in the tricarboxylic acid cycle in Synechocystis sp. PCC 6803: discovery of important factors that directly affect the conversion of oxaloacetate

The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathways in nature. Oxygenic photoautotrophic bacteria, cyanobacteria, have an unusual TCA cycle. The TCA cycle in cyanobacteria contains two unique enzymes that are not part of the TCA cycle in other organisms. In recent years, sustainable metabolite production from carbon dioxide using cyanobacteria has been looked at as a means to reduce the environmental burden of this gas. Among cyanobacteria, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) is an optimal host for sustainable metabolite production. Recently, metabolite production using the TCA cycle in Synechocystis 6803 has been carried out. Previous studies revealed that the branch point of the oxidative and reductive TCA cycles, oxaloacetate metabolism, plays a key role in metabolite production. However, the biochemical mechanisms regulating oxaloacetate metabolism in Synechocystis 6803 are poorly understood. Concentrations of oxaloacetate in Synechocystis 6803 are extremely low, such that in vivo analysis of oxaloacetate metabolism does not seem realistic. Therefore, using purified enzymes, we reconstituted oxaloacetate metabolism in Synechocystis 6803 in vitro to reveal the regulatory mechanisms involved. Reconstitution of oxaloacetate metabolism revealed that pH, Mg²⁺ and phosphoenolpyruvate are important factors affecting the conversion of oxaloacetate in the TCA cycle. Biochemical analyses of the enzymes involved in oxaloacetate metabolism in this and previous studies revealed the biochemical mechanisms underlying the effects of these factors on oxaloacetate conversion. In addition, we clarified the function of two l- malate dehydrogenase isozymes in oxaloacetate metabolism. These findings serve as a basis for various applications of the cyanobacterial TCA cycle.     

Thermodynamics of natural selection II: Chemical Carnot cycles

This is the second in a series of three papers devoted to energy flow and entropy changes in chemical and biological processes, and to their relations to the thermodynamics of computation. In the first paper of the series, it was shown that a general-form dimensional argument from the second law of thermodynamics captures a number of scaling relations governing growth and development across many domains of life. It was also argued that models of physiology based on reversible transformations provide sensible approximations within which the second-law scaling is realized. This paper provides a formal basis for decomposing general cyclic, fixed-temperature chemical reactions, in terms of the chemical equivalent of Carnot's cycle for heat engines. It is shown that the second law relates the minimal chemical work required to perform a cycle to the Kullback-Leibler divergence produced in its chemical output ensemble from that of a Gibbs equilibrium. Reversible models of physiology are used to create reversible models of natural selection, which relate metabolic energy requirements to information gain under optimal conditions. When dissipation is added to models of selection, the second-law constraint is generalized to a relation between metabolic work and the combined energies of growth and maintenance.     

The aspartate-family pathway of plants: Linking production of essential amino acids with energy and stress regulation

The Asp family pathway of plants is highly important from a nutritional standpoint because it leads to the synthesis of the four essential amino acids Lys, Thr, Met and Ile. These amino acids are not synthesized by human and its monogastric livestock and should be supplemented in their diets. Among the Asp-family amino acids, Lys is considered as the nutritionally most important essential amino acid because its level is most limiting in cereal grains, representing the largest source of plant foods and feeds worldwide. Metabolic engineering approaches led to significant increase in Lys level in seeds by enhancing its synthesis and reducing its catabolism. However, results from the model plant Arabidopsis showed that this approach may retard seed germination due to a major negative effect on the levels of a number of TCA cycle metabolites that associate with cellular energy. In the present review, we discuss the regulatory metabolic link of the Asp-family pathway with the TCA cycle and its biological significance upon exposure to stress conditions that cause energy deprivation. In addition, we also discuss how deep understanding of the regulatory metabolic link of the Asp-family pathway with energy and stress regulation can be used to improve Lys level in seeds of important crop species, minimizing the interference with the cellular energy status and plant-stress interaction. This review thus provides an example showing how deep understanding the inter-regulation of metabolism with plant stress physiology can lead to successful nutritional improvements with minimal negative effect on plant growth and response to stressful environments.Key words: Lysine, metabolic engineering, essential amino acids, plants energy, TCA cycle