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In recent decades, the role of tumor biomechanics on cancer cell behavior at the primary site has been increasingly appreciated. However, the effect of primary tumor biomechanics on the latter stages of the metastatic cascade, such as metastatic seeding of secondary sites and outgrowth remains underappreciated. This work sought to address this in the context of triple negative breast cancer (TNBC), a cancer type known to aggressively disseminate at all stages of disease progression. Using mechanically tuneable model systems, mimicking the range of stiffness's typically found within breast tumors, it is found that, contrary to expectations, cancer cells exposed to softer microenvironments are more able to colonize secondary tissues. It is shown that heightened cell survival is driven by enhanced metabolism of fatty acids within TNBC cells exposed to softer microenvironments. It is demonstrated that uncoupling cellular mechanosensing through integrin β1 blocking antibody effectively causes stiff primed TNBC cells to behave like their soft counterparts, both in vitro and in vivo. This work is the first to show that softer tumor microenvironments may be contributing to changes in disease outcome by imprinting on TNBC cells a greater metabolic flexibility and conferring discrete cell survival advantages. [ABSTRACT FROM AUTHOR]

The nicotinamide adenine dinucleotide (NAD+) precursor nicotinamide mononucleotide (NMN) is a proposed therapy for age‐related disease, whereby it is assumed that NMN is incorporated into NAD+ through the canonical recycling pathway. During oral delivery, NMN is exposed to the gut microbiome, which could modify the NAD+ metabolome through enzyme activities not present in the mammalian host. We show that orally delivered NMN can undergo deamidation and incorporation in mammalian tissue via the de novo pathway, which is reduced in animals treated with antibiotics to ablate the gut microbiome. Antibiotics increased the availability of NAD+ metabolites, suggesting the microbiome could be in competition with the host for dietary NAD+ precursors. These findings highlight new interactions between NMN and the gut microbiome. [ABSTRACT FROM AUTHOR]

Efficacy of therapies that target the downstream nitric oxide (NO) pathway in pulmonary arterial hypertension (PAH) depends on the bioavailability of NO. Reduced NO level in PAH is secondary to "uncoupling" of endothelial nitric oxide synthase (eNOS). Stimulation of β3 adrenergic receptors (β3 ARs) may lead to the recoupling of NOS and therefore be beneficial in PAH. We aimed to examine the efficacy of β3 AR agonism as a novel pathway in experimental PAH. In hypoxia (5 weeks) and Sugen hypoxia (hypoxia for 5 weeks + SU5416 injection) models of PAH, we examined the effects of the selective β3 AR agonist CL316243. We measured echocardiographic indices and invasive right ventricular (RV)–pulmonary arterial (PA) hemodynamics and compared CL316243 with riociguat and sildenafil. We assessed treatment effects on RV–PA remodeling, oxidative stress, and eNOS glutathionylation, an oxidative modification that uncouples eNOS. Compared with normoxic mice, RV systolic pressure was increased in the control hypoxic mice (p < 0.0001) and Sugen hypoxic mice (p < 0.0001). CL316243 reduced RV systolic pressure, to a similar degree to riociguat and sildenafil, in both hypoxia (p < 0.0001) and Sugen hypoxia models (p < 0.03). CL316243 reversed pulmonary vascular remodeling, decreased RV afterload, improved RV–PA coupling efficiency and reduced RV stiffness, hypertrophy, and fibrosis. Although all treatments decreased oxidative stress, CL316243 significantly reduced eNOS glutathionylation. β3 AR stimulation improved RV hemodynamics and led to beneficial RV–PA remodeling in experimental models of PAH. β3 AR agonists may be effective therapies in PAH. [ABSTRACT FROM AUTHOR]

The topology of metabolic networks is recognisably modular with modules weakly connected apart from sharing a pool of currency metabolites. Here, we defined modules as sets of reversible reactions isolated from the rest of metabolism by irreversible reactions except for the exchange of currency metabolites. Our approach identifies topologically independent modules under specific conditions associated with different metabolic functions. As case studies, the E.coli iJO1366 and Human Recon 2.2 genome-scale metabolic models were split in 103 and 321 modules respectively, displaying significant correlation patterns in expression data. Finally, we addressed a fundamental question about the metabolic flexibility conferred by reversible reactions: "Of all Directed Topologies (DTs) defined by fixing directions to all reversible reactions, how many are capable of carrying flux through all reactions?". Enumeration of the DTs for iJO1366 model was performed using an efficient depth-first search algorithm, rejecting infeasible DTs based on mass-imbalanced and loopy flux patterns. We found the direction of 79% of reversible reactions must be defined before all directions in the network can be fixed, granting a high degree of flexibility. Author summary: Genome-scale metabolic reconstructions represent all biochemical reactions that an organism can accomplish. These reconstructions are complex and often difficult to study in great detail. A way to overcome this limitation is to focus on specific pathways or subsystems. We present a novel method to identify metabolic modules based on the network topology. The method relies on reaction directions and ignores currency metabolites, which artificially connect distant metabolic reactions. In this way, topologically independent modules are built, where inputs and outputs are controlled by irreversible reactions. The method is automatic and unbiased, and, the result is a set of condition specific modules with defined metabolic functions. As a proof-of-concept we generated biologically relevant modules for the E.coli and Human genome-scale metabolic reconstructions supported by transcriptomic data. Finally, we applied the novel approach to study the network flexibility conferred by reversible reactions. In the case of the E. coli model, we found that the direction of 79% of structurally reversible reactions (those not directionally constrained by surrounding irreversible reactions) must be fixed to determine all the reaction directions in the network. Therefore, reversible reactions operate practically independent of each other. [ABSTRACT FROM AUTHOR]

Abstract Background The quantitative analysis of metabolic fluxes, i.e., in vivo activities of intracellular enzymes and pathways, provides key information on biological systems in systems biology and metabolic engineering. It is based on a comprehensive approach combining (i) tracer cultivation on 13C substrates, (ii) 13C labelling analysis by mass spectrometry and (iii) mathematical modelling for experimental design, data processing, flux calculation and statistics. Whereas the cultivation and the analytical part is fairly advanced, a lack of appropriate modelling software solutions for all modelling aspects in flux studies is limiting the application of metabolic flux analysis. Results We have developed OpenFLUX as a user friendly, yet flexible software application for small and large scale 13C metabolic flux analysis. The application is based on the new Elementary Metabolite Unit (EMU) framework, significantly enhancing computation speed for flux calculation. From simple notation of metabolic reaction networks defined in a spreadsheet, the OpenFLUX parser automatically generates MATLAB-readable metabolite and isotopomer balances, thus strongly facilitating model creation. The model can be used to perform experimental design, parameter estimation and sensitivity analysis either using the built-in gradient-based search or Monte Carlo algorithms or in user-defined algorithms. Exemplified for a microbial flux study with 71 reactions, 8 free flux parameters and mass isotopomer distribution of 10 metabolites, OpenFLUX allowed to automatically compile the EMU-based model from an Excel file containing metabolic reactions and carbon transfer mechanisms, showing it's user-friendliness. It reliably reproduced the published data and optimum flux distributions for the network under study were found quickly ( Conclusion We have developed a fast, accurate application to perform steady-state 13C metabolic flux analysis. OpenFLUX will strongly facilitate and enhance the design, calculation and interpretation of metabolic flux studies. By providing the software open source, we hope it will evolve with the rapidly growing field of fluxomics.

Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilization based on nutritional status. This is coordinated by insulin, which triggers kinase signaling cascades to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, we sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride-glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting that distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed that glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signaling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilizes protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo. Together, these data highlight the importance of glucosemetabolismto support insulin action, providing a complementary regulatory mechanismto signal transduction to stimulate adipose anabolism. [ABSTRACT FROM AUTHOR]

Abstract: Lactate is one of the key waste metabolites of mammalian cell culture. High lactate levels are caused by high aerobic glycolysis, also known as the Warburg effect, and are usually associated with adverse culture performance. Therefore, reducing lactate accumulation has been an ongoing challenge in the cell culture development to improve growth, productivity, and process robustness. The pyruvate dehydrogenase complex (PDC) plays a crucial role for the fate of pyruvate, as it converts pyruvate to acetyl coenzyme A (acetyl‐CoA). The PDC activity can be indirectly increased by inhibiting the PDC inhibitor, pyruvate dehydrogenase kinase, using dichloroacetate (DCA), resulting in less pyruvate being available for lactate formation. Here, Chinese hamster ovary cells were cultivated either with 5 mM DCA or without DCA in various batch and fed‐batch bioreactor processes. In all cultures, DCA increased peak viable cell density (VCD), culture length and final antibody titer. The strongest effect was observed in a fed batch with media and glucose feeding in which peak VCD was increased by more than 50%, culture length was extended by more than 3 days, and the final antibody titer increased by more than twofold. In cultures with DCA, lactate production and glucose consumption during exponential growth were on average reduced by approximately 40% and 35%, respectively. Metabolic flux analysis showed reduced glycolytic fluxes, whereas fluxes in the tricarboxylic acid (TCA) cycle were not affected, suggesting that cultures with DCA use glucose more efficiently. In a proteomics analysis, only few proteins were identified as being differentially expressed, indicating that DCA acts on a posttranslational level. Antibody quality in terms of aggregation, charge variant, and glycosylation pattern was unaffected. Subsequent bioreactor experiments with sodium lactate and sodium chloride feeding indicated that lower osmolality, rather than lower lactate concentration itself, improved culture performance in DCA cultures. In conclusion, the addition of DCA to the cell culture improved culture performance and increased antibody titers without any disadvantages for cell‐specific productivity or antibody quality. [ABSTRACT FROM AUTHOR]

The compartmentalization of C 4 plants increases photosynthetic efficiency, while constraining how material and energy must flow in leaf tissues. To capture this metabolic phenomenon, a generic plant metabolic reconstruction was replicated into four connected spatiotemporal compartments, namely bundle sheath (B) and mesophyll (M) across the day and night cycle. The C 4 leaf model was used to explore how amenable polyhydroxybutyrate (PHB) production is with these four compartments working cooperatively. A strategic pattern of metabolite conversion and exchange emerged from a systems-level network that has very few constraints imposed; mainly the sequential two-step carbon capture in mesophyll, then bundle sheath and photosynthesis during the day only. The building of starch reserves during the day and their mobilization during the night connects day and night metabolism. Flux simulations revealed that PHB production did not require rerouting of metabolic pathways beyond what is already utilised for growth. PHB yield was sensitive to photoassimilation capacity, availability of carbon reserves, ATP maintenance, relative photosynthetic activity of B and M, and type of metabolites exchanged in the plasmodesmata, but not sensitive towards compartmentalization. Hence, the compartmentalization issues currently encountered are likely to be kinetic or thermodynamic limitations rather than stoichiometric. [ABSTRACT FROM AUTHOR]

Pyruvate dehydrogenase (PDH) activity is a key component of the glucose/fatty acid cycle hypothesis for the regulation of glucose uptake and metabolism. We have investigated whether acute activation of PDH in muscle can alleviate the insulin resistance caused by feeding animals a high-fat diet (HFD). The importance of PDH activity in muscle glucose disposal under insulin-stimulated conditions was determined by infusing the PDH kinase inhibitor dichloroacetate (DCA) into HFD-fed Wistar rats during a hyperinsulinemiceuglycemic clamp. Acute DCA infusion did not alter glucose infusion rate, glucose disappearance, or hepatic glucose production but did decrease plasma lactate levels. DCA substantially increased muscle PDH activity; however, this did not improve insulin-stimulated glucose uptake in insulin-resistant muscle of HFD rats. DCA infusion increased the flux of pyruvate to acetyl-CoA and reduced glucose incorporation into glycogen and alanine in muscle. Similarly, in isolated muscle, DCA treatment increased glucose oxidation and decreased glycogen synthesis without changing glucose uptake. These results suggest that, although PDH activity controls the conversion of pyruvate to acetyl-CoA for oxidation, this has little effect on glucose uptake into muscle under insulin-stimulated conditions. [ABSTRACT FROM AUTHOR]

Skeletal muscle is a major tissue for glucose metabolism and can store glucose as glycogen, convert glucose to lactate via glycolysis and fully oxidise glucose to CO2. Muscle has a limited capacity for gluconeogenesis but can convert lactate and alanine to glycogen. Gluconeogenesis requires FBP2, a muscle-specific form of fructose bisphosphatase that converts fructose-1,6-bisphosphate (F-1,6-bisP) to fructose-6-phosphate (F-6-P) opposing the activity of the ATP-consuming enzyme phosphofructokinase (PFK). In mammalian muscle, the activity of PFK is normally 100 times higher than FBP2 and therefore energy wasting cycling between PFK and FBP2 is low. In an attempt to increase substrate cycling between F-6-P and F-1,6-bisP and alter glucose metabolism, we overexpressed FBP2 using a muscle-specific adeno-associated virus (AAV-tMCK-FBP2). AAV was injected into the right tibialis muscle of rats, while the control contralateral left tibialis received a saline injection. Rats were fed a chow or 45% fat diet (HFD) for 5 weeks after which, hyperinsulinaemic-euglycaemic clamps were performed. Infection of the right tibialis with AAV-tMCK-FBP2 increased FBP2 activity 10 fold on average in chow and HFD rats (P < 0.0001). Overexpression of FBP2 significantly increased insulin-stimulated glucose uptake in tibialis of chow animals (control 14.3 ± 1.7; FBP2 17.6 ± 1.6 µmol/min/100 g) and HFD animals (control 9.6 ± 1.1; FBP2 11.2 ± 1.1µmol/min/100 g). The results suggest that increasing the capacity for cycling between F-1,6-bisP and F-6-P can increase the metabolism of glucose by introducing a futile cycle in muscle, but this increase is not sufficient to overcome muscle insulin resistance. [ABSTRACT FROM AUTHOR]

Insulin triggers an extensive signaling cascade to coordinate adipocyte glucose metabolism. It is considered that the major role of insulin is to provideanabolic substrates by activating GLUT4-dependent glucose uptake. However, insulin stimulates phosphorylation of many metabolic proteins. To examine the implications of this on glucose metabolism, we performed dynamic tracer metabolomics in cultured adipocytes treated with insulin. Temporal analysis of metabolite concentrations and tracer labeling revealed rapid and distinct changes in glucose metabolism, favoring specific glycolytic branch points and pyruvate anaplerosis. Integrating dynamic metabolomics and phosphoproteomics data revealed that insulindependent phosphorylation of anabolic enzymes occurred prior to substrate accumulation. Indeed, glycogen synthesis was activated independently of glucose supply. We refer to this phenomenon as metabolic priming, whereby insulin signaling creates a demand-driven system to ''pull'' glucose into specific anabolic pathways. This complements the supply- driven regulation of anabolism by substrate accumulation and highlights an additional role for insulin action in adipocyte glucose metabolism. [ABSTRACT FROM AUTHOR]

Gas fermentation using acetogenic bacteria offers a promising route for the sustainable production of low carbon fuels and commodity chemicals from abundant, inexpensive C1 feedstocks including industrial waste gases, syngas, reformed methane or methanol. Clostridium autoethanogenum is a model gas fermenting acetogen that produces fuel ethanol and 2,3-butanediol, a precursor for nylon and rubber. Acetogens have already been used in large scale industrial fermentations, they are ubiquitous and known to play a prominent role in the global carbon cycle. Still, they are considered to live on the thermodynamic edge of life and potential energy constraints when growing on C1 gases pose a major challange for the commercial production of fuels and chemicals. We have developed a systematic platform to investigate acetogenic energy metabolism, exemplified here by experiments contrasting heterotrophic and autotrophic metabolism. The platform is built from complete omics technologies, augmented with genetic tools and complemented by a manually curated genome-scale mathematical model. Together the tools enable the design and development of new, energy efficient pathways and strains for the production of chemicals and advanced fuels via C1 gas fermentation. As a proof-of-platform, we investigated heterotrophic growth on fructose versus autotrophic growth on gas that demonstrate the role of the Rnf complex and Nfn complex in maintaining growth using the Wood–Ljungdahl pathway. Pyruvate carboxykinase was found to control the rate-limiting step of gluconeogenesis and a new specialized glyceraldehyde-3-phosphate dehydrogenase was identified that potentially enhances anabolic capacity by reducing the amount of ATP consumed by gluconeogenesis. The results have been confirmed by the construction of mutant strains. [ABSTRACT FROM AUTHOR]

Genome scale metabolic modeling has traditionally been used to explore metabolism of individual cells or tissues. In higher organisms, the metabolism of individual tissues and organs is coordinated for the overall growth and well-being of the organism. Understanding the dependencies and rationale for multicellular metabolism is far from trivial. Here, we have advanced the use of AraGEM (a genome-scale reconstruction of Arabidopsis metabolism) in a multi-tissue context to understand how plants grow utilizing their leaf, stem and root systems across the day-night (diurnal) cycle. Six tissue compartments were created, each with their own distinct set of metabolic capabilities, and hence a reliance on other compartments for support. We used the multi-tissue framework to explore differences in the "division-of-labor" between the sources and sink tissues in response to: (a) the energy demand for the translocation of C and N species in between tissues; and (b) the use of two distinct nitrogen sources (NO3- or NH4+). The "division-of-labor" between compartments was investigated using a minimum energy (photon) objective function. Random sampling of the solution space was used to explore the flux distributions under different scenarios as well as to identify highly coupled reaction sets in different tissues and organelles. Efficient identification of these sets was achieved by casting this problem as a maximum clique enumeration problem. The framework also enabled assessing the impact of energetic constraints in resource (redox and ATP) allocation between leaf, stem, and root tissues required for efficient carbon and nitrogen assimilation, including the diurnal cycle constraint forcing the plant to set aside resources during the day and defer metabolic processes that are more efficiently performed at night. This study is a first step toward autonomous modeling of whole plant metabolism. [ABSTRACT FROM AUTHOR]

As human embryonic stem cells (hESCs) steadily progress towards regenerative medicine applications there is an increasing emphasis on the development of bioreactor platforms that enable expansion of these cells to clinically relevant numbers. Surprisingly little is known about the metabolic requirements of hESCs, precluding the rational design and optimisation of such platforms. In this study, we undertook an in-depth characterisation of MEL-2 hESC metabolic behaviour during the exponential growth phase, combining metabolic profiling and flux analysis tools at physiological (hypoxic) and atmospheric (normoxic) oxygen concentrations. To overcome variability in growth profiles and the problem of closing mass balances in a complex environment, we developed protocols to accurately measure uptake and production rates of metabolites, cell density, growth rate and biomass composition, and designed a metabolic flux analysis model for estimating internal rates. hESCs are commonly considered to be highly glycolytic with inactive or immature mitochondria, however, whilst the results of this study confirmed that glycolysis is indeed highly active, we show that at least in MEL-2 hESC, it is supported by the use of oxidative phosphorylation within the mitochondria utilising carbon sources, such as glutamine to maximise ATP production. Under both conditions, glycolysis was disconnected from the mitochondria with all of the glucose being converted to lactate. No difference in the growth rates of cells cultured under physiological or atmospheric oxygen concentrations was observed nor did this cause differences in fluxes through the majority of the internal metabolic pathways associated with biogenesis. These results suggest that hESCs display the conventional Warburg effect, with high aerobic activity despite high lactate production, challenging the idea of an anaerobic metabolism with low mitochondrial activity. The results of this study provide new insight that can be used in rational bioreactor design and in the development of novel culture media for hESC maintenance and expansion. [ABSTRACT FROM AUTHOR]

Mammalian cell cultures typically exhibit an energy inefficient phenotype characterized by the consumption of large quantities of glucose and the concomitant production of large quantities of lactate. Under certain conditions, mammalian cells can switch to a more energy efficient state during which lactate is consumed. Using a metabolic model derived from a mouse genome scale model we performed flux balance analysis of Chinese hamster ovary cells before and after a metabolic switch from lactate production (in the presence of glucose) to lactate consumption (after glucose depletion). Despite a residual degree of freedom after accounting for measurements, the calculated flux ranges and associated errors were narrow enough to enable investigation of metabolic changes across the metabolic switch. Surprisingly, the fluxes through the lower part of the TCA cycle from oxoglutarate to malate were very similar (around 60 µmol/gDW/h) for both phases. A detailed analysis of the energy metabolism showed that cells consuming lactate have an energy efficiency (total ATP produced per total C-mol substrate consumed) six times greater than lactate producing cells. Biotechnol. Bioeng. 2013; 110: 660-666. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Mammalian cell cultures are the predominant system for the production of recombinant proteins requiring post-translational modifications. As protein yields are a function of growth performance (among others), and performance varies greatly between culture medium (e.g., different growth rates and peak cell densities), an understanding of the biological mechanisms underpinning this variability would facilitate rational medium and process optimization, increasing product yields, and reducing costs. We employed a metabolomics approach to analyze differences in metabolite concentrations of CHO cells cultivated in three different media exhibiting different growth rates and maximum viable cell densities. Analysis of intra- and extracellular metabolite concentrations over the course of the cultures using a combination of HPLC and GC-MS, readily detected medium specific and time dependent changes. Using multivariate data analysis, we identified a range of metabolites correlating with growth rate, illustrating how metabolomics can be used to relate gross phenotypic changes to the fine details of cellular metabolism. Biotechnol. Bioeng. 2012; 109:1404-1414. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: Microalgae have the potential to deliver biofuels without the associated competition for land resources. In order to realise the rates and titres necessary for commercial production, however, system-level metabolic engineering will be required. Genome scale metabolic reconstructions have revolutionized microbial metabolic engineering and are used routinely for in silico analysis and design. While genome scale metabolic reconstructions have been developed for many prokaryotes and model eukaryotes, the application to less well characterized eukaryotes such as algae is challenging not at least due to a lack of compartmentalization data. Results: We have developed a genome-scale metabolic network model (named AlgaGEM) covering the metabolism for a compartmentalized algae cell based on the Chlamydomonas reinhardtii genome. AlgaGEM is a comprehensive literature-based genome scale metabolic reconstruction that accounts for the functions of 866 unique ORFs, 1862 metabolites, 2249 gene-enzyme-reaction-association entries, and 1725 unique reactions. The reconstruction was compartmentalized into the cytoplasm, mitochondrion, plastid and microbody using available data for algae complemented with compartmentalisation data for Arabidopsis thaliana. AlgaGEM describes a functional primary metabolism of Chlamydomonas and significantly predicts distinct algal behaviours such as the catabolism or secretion rather than recycling of phosphoglycolate in photorespiration. AlgaGEM was validated through the simulation of growth and algae metabolic functions inferred from literature. Using efficient resource utilisation as the optimality criterion, AlgaGEM predicted observed metabolic effects under autotrophic, heterotrophic and mixotrophic conditions. AlgaGEM predicts increased hydrogen production when cyclic electron flow is disrupted as seen in a high producing mutant derived from mutational studies. The model also predicted the physiological pathway for H2 production and identified new targets to further improve H2 yield. Conclusions: AlgaGEM is a viable and comprehensive framework for in silico functional analysis and can be used to derive new, non-trivial hypotheses for exploring this metabolically versatile organism. Flux balance analysis can be used to identify bottlenecks and new targets to metabolically engineer microalgae for production of biofuels. [ABSTRACT FROM AUTHOR]

Leaves of C4 grasses (such as maize [Zea mays], sugarcane [Saccharum officinarum], and sorghum [Sorghum bicolor]) form a classical Kranz leaf anatomy. Unlike C3 plants, where photosynthetic CO2 fixation proceeds in the mesophyll (M), the fixation process in C4 plants is distributed between two cell types, the M cell and the bundle sheath (BS) cell. Here, we develop a C4 genome-scale model (C4GEM) for the investigation of flux distribution in M and BS cells during C4 photosynthesis. C4GEM, to our knowledge, is the first large-scale metabolic model that encapsulates metabolic interactions between two different cell types. C4GEM is based on the Arabidopsis (Arabidopsis thaliana) model (AraGEM) but has been extended by adding reactions and transporters responsible to represent three different C4 subtypes (NADP-ME [for malic enzyme], NAD-ME, and phosphoenolpyruvate carboxykinase). C4GEM has been validated for its ability to synthesize 47 biomass components and consists of 1,588 unique reactions, 1,755 metabolites, 83 interorganelle transporters, and 29 external transporters (including transport through plasmodesmata). Reactions in the common C4 model have been associated with well-annotated C4 species (NADP-ME subtypes): 3,557 genes in sorghum, 11,623 genes in maize, and 3,881 genes in sugarcane. The number of essential reactions not assigned to genes is 131,135, and 156 in sorghum, maize, and sugarcane, respectively. Flux balance analysis was used to assess the metabolic activity in M and BS cells during C4 photosynthesis. Our simulations were consistent with chloroplast proteomic studies, and C4GEM predicted the classical C4 photosynthesis pathway and its major effect in organelle function in M and BS. The model also highlights differences in metabolic activities around photosystem I and photosystem II for three different C4 subtypes. Effects of CO2 leakage were also explored. C4GEM is a viable framework for in silico analysis of cell cooperation between M and BS cells during photosynthesis and can be used to explore C4 plant metabolism. [ABSTRACT FROM AUTHOR]

Background: The quantitative analysis of metabolic fluxes, i.e., in vivo activities of intracellular enzymes and pathways, provides key information on biological systems in systems biology and metabolic engineering. It is based on a comprehensive approach combining (i) tracer cultivation on 13C substrates, (ii) 13C labelling analysis by mass spectrometry and (iii) mathematical modelling for experimental design, data processing, flux calculation and statistics. Whereas the cultivation and the analytical part is fairly advanced, a lack of appropriate modelling software solutions for all modelling aspects in flux studies is limiting the application of metabolic flux analysis. Results: We have developed OpenFLUX as a user friendly, yet flexible software application for small and large scale 13C metabolic flux analysis. The application is based on the new Elementary Metabolite Unit (EMU) framework, significantly enhancing computation speed for flux calculation. From simple notation of metabolic reaction networks defined in a spreadsheet, the OpenFLUX parser automatically generates MATLAB-readable metabolite and isotopomer balances, thus strongly facilitating model creation. The model can be used to perform experimental design, parameter estimation and sensitivity analysis either using the built-in gradient-based search or Monte Carlo algorithms or in user-defined algorithms. Exemplified for a microbial flux study with 71 reactions, 8 free flux parameters and mass isotopomer distribution of 10 metabolites, OpenFLUX allowed to automatically compile the EMU-based model from an Excel file containing metabolic reactions and carbon transfer mechanisms, showing it's user-friendliness. It reliably reproduced the published data and optimum flux distributions for the network under study were found quickly (<20 sec). Conclusion: We have developed a fast, accurate application to perform steady-state 13C metabolic flux analysis. OpenFLUX will strongly facilitate and enhance the design, calculation and interpretation of metabolic flux studies. By providing the software open source, we hope it will evolve with the rapidly growing field of fluxomics. [ABSTRACT FROM AUTHOR]

The zinc finger transcription factor Snail is a known effector of epithelial-to-mesenchymal transition (EMT), a process that underlies the enhanced invasiveness and chemoresistance of common to cancerous cells. Induction of Snail-driven EMT has also been shown to drive a range of pro-survival metabolic adaptations in different cancers. In the present study, we sought to determine the specific role that Snail has in driving EMT and adaptive metabolic programming in pancreatic ductal adenocarcinoma (PDAC) by overexpressing Snail in a PDAC cell line, Panc1, and in immortalized, non-tumorigenic human pancreatic ductal epithelial (HPDE) cells. Snail overexpression was able to induce EMT in both pancreatic cell lines through suppression of epithelial markers and upregulation of mesenchymal markers alongside changes in cell morphology and enhanced migratory capacity. Snail-overexpressed pancreatic cells additionally displayed increased glucose uptake and lactate production with concomitant reduction in oxidative metabolism measurements. Snail overexpression reduced maximal respiration in both Panc1 and HPDE cells, with further reductions seen in ATP production, spare respiratory capacity and non-mitochondrial respiration in Snail overexpressing Panc1 cells. Accordingly, lower expression of mitochondrial electron transport chain proteins was observed with Snail overexpression, particularly within Panc1 cells. Modelling of 13C metabolite flux within both cell lines revealed decreased carbon flux from glucose in the TCA cycle in snai1-overexpressing Panc1 cells only. This work further highlights the role that Snail plays in EMT and demonstrates its specific effects on metabolic reprogramming of glucose metabolism in PDAC. [ABSTRACT FROM AUTHOR]

A small minority (< 3%) of protein-coding genetic variants are predicted to lead to loss of protein function. However, these predicted loss-of-function (pLOF) variants can provide insight into mode of transcriptional effect. To examine how these changes are propagated to phenotype, we determined associations with downstream metabolites. We performed association analyses of 37 pLOF variants – previously reported to be significantly associated with disease in >400,000 subjects in UK Biobank – with metabolites. We conducted these analyses in three community-based cohorts: the Framingham Heart Study (FHS) Offspring Cohort, FHS Generation 3, and the KORA F4 cohort. We identified 19 new low-frequency or rare (minor allele frequency (MAF) <5%) pLOF variant-metabolite associations, and 12 new common (MAF > 5%) pLOF variant-metabolite associations. Rare pLOF variants in the genes BTN3A2 , ENPEP, and GEM that have been associated with blood pressure in UK Biobank, were associated with vasoactive metabolites indoxyl sulfate, asymmetric dimethylarginine (ADMA), and with niacinamide, respectively. A common pLOF variant in gene CCHCR1 , associated with asthma in UK Biobank, was associated with histamine and niacinamide in FHS Generation 3, both reported to play a role in this disease. Common variants in olfactory receptor gene OX4C11 that associated with blood pressure in UK Biobank were associated with the nicotine metabolite cotinine, suggesting an interaction between altered olfaction, smoking behaviour, and blood pressure. These findings provide biological validity for pLOF variant-disease associations, and point to the effector roles of common metabolites. Such an approach may provide novel disease markers and therapeutic targets. [ABSTRACT FROM AUTHOR]

Sirtuins are a family of evolutionary conserved enzymes that dynamically regulate cellular physiology. Mammals have 7 sirtuins, which are located in different cellular compartments. Sirt5, a sirtuin isoform located in multiple subcellular sites, is involved in regulating a diverse range of cellular and metabolic processes through the removal of a range of acyl-lysine modifications on target proteins. Loss of Sirt5 leads to hyper-malonylation and hyper-succinylation of both mitochondrial and extra-mitochondrial proteins, influencing oxidative phosphorylation, the TCA cycle and glycolysis. However despite these findings, the effect of Sirt5 overexpression on metabolism remains poorly investigated. Here we report that overexpression of Sirt5 has minimal effect on mitochondrial metabolism and overall physiology in mice, despite inducing widespread decreases in protein acylation. Our data confirms the role of Sirt5 as an important demalonylase and desuccinylase enzyme in vivo , but questions the relevance of physiological changes in protein acylation levels in the regulation of cellular metabolism. [ABSTRACT FROM AUTHOR]

Solid tumors are highly reliant on lipids for energy, growth, and survival. In prostate cancer, the activity of the androgen receptor (AR) is associated with reprogramming of lipid metabolic processes. Here, we identified acyl-CoA synthetase medium chain family members 1 and 3 (ACSM1 and ACSM3) as AR-regulated mediators of prostate cancer metabolism and growth. ACSM1 and ACSM3 were upregulated in prostate tumors compared with nonmalignant tissues and other cancer types. Both enzymes enhanced proliferation and protected prostate cancer cells from death in vitro, whereas silencing ACSM3 led to reduced tumor growth in an orthotopic xenograft model. ACSM1 and ACSM3 were major regulators of the prostate cancer lipidome and enhanced energy production via fatty acid oxidation. Metabolic dysregulation caused by loss of ACSM1/3 led to mitochondrial oxidative stress, lipid peroxidation, and cell death by ferroptosis. Conversely, elevated ACSM1/3 activity enabled prostate cancer cells to survive toxic levels of medium chain fatty acids and promoted resistance to ferroptosis-inducing drugs and AR antagonists. Collectively, this study reveals a tumor-promoting function of medium chain acyl-CoA synthetases and positions ACSM1 and ACSM3 as key players in prostate cancer progression and therapy resistance. Significance: Androgen receptor-induced ACSM1 and ACSM3 mediate a metabolic pathway in prostate cancer that enables the utilization of medium chain fatty acids for energy production, blocks ferroptosis, and drives resistance to clinically approved antiandrogens., (©2024 American Association for Cancer Research.)

Almost 40% of adults worldwide are classified as overweight or obese. Exercise is a beneficial intervention in obesity, partly due to increases in mitochondrial activity and subsequent increases in nicotinamide adenine dinucleotide (NAD + ), an important metabolic cofactor. Recent studies have shown that increasing NAD + levels through pharmacological supplementation with precursors such as nicotinamide mononucleotide (NMN) improved metabolic health in high-fat-diet (HFD)-fed mice. However, the effects of combined exercise and NMN supplementation are unknown. Thus, here we examined the combined effects of NMN and treadmill exercise in female mice with established obesity after 10 wk of diet. Five-week-old female C57BL/6J mice were exposed to a control diet ( n = 16) or HFD. Mice fed a HFD were either untreated (HFD; n = 16), received NMN in drinking water (400 mg/kg; HNMN; n = 16), were exposed to treadmill exercise 6 days/wk (HEx; n = 16), or were exposed to exercise combined with NMN (HNEx; n = 16). Although some metabolic benefits of NMN have been described, at this dose, NMN administration impaired several aspects of exercise-induced benefits in obese mice, including glucose tolerance, glucose-stimulated insulin secretion from islets, and hepatic triglyceride accumulation. HNEx mice also exhibited increased antioxidant and reduced prooxidant gene expression in both islets and muscle, suggesting that altered redox status is associated with the loss of exercise-induced health benefits with NMN cotreatment. Our data show that NMN treatment impedes the beneficial metabolic effects of exercise in a mouse model of diet-induced obesity in association with disturbances in redox metabolism. NEW & NOTEWORTHY NMN dampened exercise-induced benefits on glucose handling in diet-induced obesity. NMN administration alongside treadmill exercise enhanced the ratio of antioxidants to prooxidants. We suggest that NMN administration may not be beneficial when NAD + levels are replete.

Key Points: Acute nicotinamide riboside (NR) supplementation does not alter substrate metabolism at rest, during or in recovery from endurance exercise. NR does not alter NAD + -sensitive signalling pathways in human skeletal muscle. NR supplementation and acute exercise influence the NAD + metabolome., Abstract: Oral supplementation of the NAD + precursor nicotinamide riboside (NR) has been reported to alter metabolism alongside increasing sirtuin (SIRT) signalling and mitochondrial biogenesis in rodent skeletal muscle. However, whether NR supplementation can elicit a similar response in human skeletal muscle is unclear. This study assessed the effect of 7-day NR supplementation on whole-body metabolism and exercise-induced mitochondrial biogenic signalling in skeletal muscle. Eight male participants (age: 23 ± 4 years, V ̇ O 2 peak 46.5 ± 4.4 ml kg -1  min -1 ) received 1 week of NR or cellulose placebo (PLA) supplementation (1000 mg day -1 ). Muscle biopsies were collected from the medial vastus lateralis prior to supplementation and pre-, immediately post- and 3 h post-exercise (1 h of 60% W max cycling) performed following the supplementation period. There was no effect of NR supplementation on substrate utilisation at rest or during exercise or on skeletal muscle mitochondrial respiration. Global acetylation, auto-PARylation of poly ADP-ribose polymerase 1 (PARP1), acetylation of Tumour protein 53 (p53) Lys382 and Manganese superoxide dismutase (MnSOD) Lys122 were also unaffected by NR supplementation or exercise. NR supplementation did not increase skeletal muscle NAD + concentration, but it did increase the concentration of deaminated NAD + precursors nicotinic acid riboside (NAR) and nicotinic acid mononucleotide (NAM) and methylated nicotinamide breakdown products (Me2PY and Me4PY), demonstrating the skeletal muscle bioavailability of NR supplementation. In summary, 1 week of NR supplementation does not alter whole-body metabolism or skeletal muscle signal transduction pathways implicated in the mitochondrial adaptation to endurance exercise., (© 2021 The Authors. The Journal of Physiology © 2021 The Physiological Society.)

Adipose tissue is essential for whole-body glucose homeostasis, with a primary role in lipid storage. It has been previously observed that lactate production is also an important metabolic feature of adipocytes, but its relationship to adipose and whole-body glucose disposal remains unclear. Therefore, using a combination of metabolic labeling techniques, here we closely examined lactate production of cultured and primary mammalian adipocytes. Insulin treatment increased glucose uptake and conversion to lactate, with the latter responding more to insulin than did other metabolic fates of glucose. However, lactate production did not just serve as a mechanism to dispose of excess glucose, because we also observed that lactate production in adipocytes did not solely depend on glucose availability and even occurred independently of glucose metabolism. This suggests that lactate production is prioritized in adipocytes. Furthermore, knocking down lactate dehydrogenase specifically in the fat body of Drosophila flies lowered circulating lactate and improved whole-body glucose disposal. These results emphasize that lactate production is an additional metabolic role of adipose tissue beyond lipid storage and release., (© 2020 Krycer et al.)