Mammalian Cell Metabolism Research Articles

Chapter Eighteen - Real-Time Assessment of the Metabolic Profile of Living Cells with Genetically Encoded NADH Sensors

Redox metabolism plays a critical role in multiple pathophysiological settings, including oncogenesis and tumor progression. Until recently, however, our knowledge of key redox processes in living systems was limited by the lack of an adequate methodology to monitor redox potential. Nicotinamide adenine dinucleotide, in its reduced (NADH) and oxidized (NAD(+)) forms, is perhaps the most important small molecule in the redox metabolism of mammalian cells. We have previously developed a series of genetically encoded fluorescent sensors allowing for the quantification of intracellular NADH. Here, we present experimental components and considerations that are required to perform a standardized quantification of intracellular NADH based on these probes. Moreover, we present the initial calibration experiments necessary to obtain reliable data from this approach, we detail a protocol to measure intracellular NADH levels in steady-state kinetic experiments, and we provide consideration on the processing of data. Among various applications, this technique is suitable for the study of redox alterations in malignant cells.

Cytochrome P450 CYP1B1 Interacts with 8-Methoxypsoralen (8-MOP) and Influences Psoralen-Ultraviolet A (PUVA) Sensitivity

BackgroundThere are unpredictable inter-individual differences in sensitivity to psoralen-UVA (PUVA) photochemotherapy, used to treat skin diseases including psoriasis. Psoralens are metabolised by cytochrome P450 enzymes (P450), and we hypothesised that variability in cutaneous P450 expression may influence PUVA sensitivity. We previously showed that P450 CYP1B1 was abundantly expressed in human skin and regulated by PUVA, and described marked inter-individual differences in cutaneous CYP1B1 expression.ObjectivesWe investigated whether CYP1B1 made a significant contribution to 8-methoxypsoralen (8-MOP) metabolism, and whether individuality in CYP1B1 activity influenced PUVA sensitivity.MethodsWe used E. coli membranes co-expressing various P450s and cytochrome P450 reductase (CPR) to study 8-MOP metabolism and cytotoxicity assays in CYP1B1-expressing mammalian cells to assess PUVA sensitivity.ResultsWe showed that P450s CYP1A1, CYP1A2, CYP1B1, CYP2A6 and CYP2E1 influence 8-MOP metabolism. As CYP1B1 is the most abundant P450 in human skin, we further demonstrated that: (i) CYP1B1 interacts with 8-MOP (ii) metabolism of the CYP1B1 substrates 7-ethoxyresorufin and 17-β-estradiol showed concentration-dependent inhibition by 8-MOP and (iii) inhibition of 7-ethoxyresorufin metabolism by 8-MOP was influenced by CYP1B1 genotype. The influence of CYP1B1 on PUVA cytotoxicity was further investigated in a Chinese hamster ovary cell line, stably expressing CYP1B1 and CPR, which was more sensitive to PUVA than control cells, suggesting that CYP1B1 metabolises 8-MOP to a more phototoxic metabolite(s).ConclusionOur data therefore suggest that CYP1B1 significantly contributes to cutaneous 8-MOP metabolism, and that individuality in CYP1B1 expression may influence PUVA sensitivity.

Overexpression of mitofusin 2 improves translocation of glucose transporter 4 in skeletal muscle of high-fat diet-fed rats through AMP-activated protein kinase signaling

Mitofusin 2 (Mfn2) is a mitochondrial membrane protein that plays a role in mitochondrial fusion and metabolism in mammalian cells. Previous studies have reported a positive correlation between Mfn2 expression and insulin sensitivity in non‑diabetic and type 2 diabetic subjects. Thus, the aim of the present study was to investigate whether Mfn2 overexpression improves insulin sensitivity of high‑fat diet (HFD) rats and the possible underlying mechanisms. Male SD rats were randomly divided into four groups: negative control; HFD; HFD plus adenoviral vectors; and HFD plus adenoviral vectors encoding Mfn2. Following an 11‑week treatment protocol, the euglycemic‑hyperinsulinemic clamp technique was applied to evaluate insulin sensitivity in rats. The skeletal muscles from rats in each group were analyzed by real‑time PCR and western blot analysis to determine glucose transporter 4 (GLUT4) expression, translocation and relative translocation signaling. Consistent with Mfn2 repression and glucose intolerance, HFD downregulates GLUT4 expression at the mRNA and protein levels, while Mfn2 overexpression activates AMP‑activated protein kinase (AMPK), increases GLUT4 expression and translocation and improves insulin resistance in the skeletal muscles of HFD rats. Results of the present study indicate that Mfn2 overexpression improves insulin sensitivity and may regulate GLUT4 translocation in an AMPK‑dependent manner in the skeletal muscles of HFD rats. This study is likely to provide insight into the unique role of Mfn2 in promoting glucose uptake, leading to modulation of GLUT4 translocation signaling and maintenance of glucose homeostasis in vivo.

Application of hybrid cybernetic model in simulating myeloma cell culture co-consuming glucose and glutamine with mixed consumption patterns

A dynamic model called hybrid cybernetic model (HCM) based on structured metabolic network is established for simulating mammalian cell metabolism featured with partially substitutable and partially complementary consumption patterns of two substrates, glucose and glutamine. Benefiting from the application of elementary mode analysis (EMA), the complicated metabolic network is decomposed into elementary modes (EMs) facilitating the employment of the hybrid cybernetic framework to investigate the external and internal flux distribution and the regulation mechanism among them. According to different substrate combination, two groups of EMs are obtained, i.e., EMs associated with glucose uptake and simultaneous uptake of glucose and glutamine. Uptake fluxes through various EMs are coupled together via cybernetic variables to maximize substrate uptake. External fluxes and internal fluxes could be calculated and estimated respectively, by the combination of the stoichiometrics of metabolic networks and fluxes through regulated EMs. The model performance is well validated via three sets of experimental data. Through parameter identification of limited number of experimental data, other external metabolites are precisely predicted. The obtained kinetic parameters of three experimental cultures have similar values, which indicates the robustness of the model. Furthermore, the prediction performance of the model is successfully validated based on identified parameters.

Effects of moderate and deep hypothermia on RNA-binding proteins RBM3 and CIRP expressions in murine hippocampal brain slices

Therapeutic hypothermia has emerged as an effective neuroprotective therapy for cardiac arrest survivors. There are a number of purported mechanisms for therapeutic hypothermia, but the exact mechanism still remains to be elucidated. Although hypothermia generally down-regulates protein synthesis and metabolism in mammalian cells, a small subset of homologous (>70%) cold-shock proteins (RNA-binding motif protein 3, RBM3 and cold-inducible RNA-binding protein, CIRP) are induced under these conditions. In addition, RBM3 up-regulation in neuronal cells has recently been implicated in hypothermia-induced neuroprotection. Therefore, we compared the effects of moderate (33.5°C) and deep (17°C) hypothermia with normothermia (37°C) on the regulation of RBM3 and CIRP expressions in murine organotypic hippocampal slice cultures (OHSC), hippocampal neuronal cells (HT-22), and microglia cells (BV-2).Moderate hypothermia resulted in significant up-regulation of both RBM3 and CIRP mRNA in murine OHSC, but deep hyporthermia did not. RBM3 protein regulation was also significantly up-regulated by 33.5°C, but no significant up-regulation of CIRP protein was observed in the OHSC. Additionally, OHSC exposed to 17°C for 24h were positive for Propidium Iodide (PI) immunostaining, indicating the onset of cell death. Similarly, RBM3 gene expression in a HT-22 neuronal cells mono-culture and direct co-culture of HT-22 neuronal cells with BV-2 microglia cells were also up-regulated at 33.5°C but only in the co-culture at 17°C. No significant up-regulation of RBM3 nor CIRP gene expression were observed in a BV-2 mono-culture at either temperature.We observed that RBM3 mRNA and protein expressions in murine OHSC, as well as in mono-culture of HT-22 neuronal cells and direct co-culture of HT-22 neuronal cells with BV-2 microglia cells were significantly up-regulated by exposure to moderate hypothermia. These findings further support the implication of RBM3 as a potential effector for hypothermia-induced neuroprotection.

Probing the metabolism of an inducible mammalian expression system using extracellular isotopomer analysis

In an effort to quantitatively assess the impact of recombinant protein expression on the primary metabolism of mammalian cells in culture, we have employed an efficient inducible expression system and conducted a comparative study of the intracellular flux map distribution with and without the induction of recombinant protein synthesis. Cells were grown in parallel semi-continuous cultures with various singly and uniformly labeled substrates and the resulting mass isotopomer distributions of lactate and extracellular amino acids were measured by mass spectrometry. These distributions were used to quantify the main intracellular fluxes. The analysis revealed that, under mild hypothermic conditions, the onset of protein expression is correlated with small but significant changes in several key pathways related to ATP and NADPH formation. More specifically, we observed that induced cells exhibit a more efficient utilization of glucose, characterized by an increased flux of pyruvate into the TCA cycle. In contrast, the catabolic rates of most amino acids remained relatively unaffected. Such analysis is instrumental to guide the identification of robust biomarkers of productivity, as well as the development of medium formulations optimized for recombinant protein production.

Metabolic host responses to infection by intracellular bacterial pathogens

The interaction of bacterial pathogens with mammalian hosts leads to a variety of physiological responses of the interacting partners aimed at an adaptation to the new situation. These responses include multiple metabolic changes in the affected host cells which are most obvious when the pathogen replicates within host cells as in case of intracellular bacterial pathogens. While the pathogen tries to deprive nutrients from the host cell, the host cell in return takes various metabolic countermeasures against the nutrient theft. During this conflicting interaction, the pathogen triggers metabolic host cell responses by means of common cell envelope components and specific virulence-associated factors. These host reactions generally promote replication of the pathogen. There is growing evidence that pathogen-specific factors may interfere in different ways with the complex regulatory network that controls the carbon and nitrogen metabolism of mammalian cells. The host cell defense answers include general metabolic reactions, like the generation of oxygen- and/or nitrogen-reactive species, and more specific measures aimed to prevent access to essential nutrients for the respective pathogen. Accurate results on metabolic host cell responses are often hampered by the use of cancer cell lines that already exhibit various de-regulated reactions in the primary carbon metabolism. Hence, there is an urgent need for cellular models that more closely reflect the in vivo infection conditions. The exact knowledge of the metabolic host cell responses may provide new interesting concepts for antibacterial therapies.

Identification ofN‐acyl‐fumonisinB1 as new cytotoxic metabolites of fumonisin mycotoxins

Fumonisins are mycotoxins produced by Fusarium species. The predominant derivative, fumonisin B1 (FB1), occurs in food and feed and is of health concern due to its hepatotoxic and carcinogenic effects. However, the role of FB1 metabolites on the mechanism of the toxicity, the inhibition of the ceramide synthesis, is unknown. The aim of this study was to identify new fumonisin metabolites and to evaluate their cytotoxic potential. MS, molecular biology, and in vitro enzyme assays were used to investigate fumonisin metabolism in mammalian cells overexpressing human ceramide synthase (CerS) genes. N-acyl-FB1 derivatives were detected as new metabolites in cultured cells at levels of up to 10 pmol/mg of protein. The N-acylation of FB1 and hydrolyzed FB1 was analyzed in several cell lines, including cells overexpressing CerS. The acyl-chain length of the N-acyl fumonisins depends on the CerS isoform acylating them. The N-acyl fumonisins are more cytotoxic than the parent fumonisin B1. The identification of N-acyl fumonisins with various acyl chain lengths together with the observed cytotoxicity of these compounds is a new aspect of fumonisin-related toxicity. Therefore, these new metabolites might play an important role in the mode of action of fumonisins.

Expanding the concepts and tools of metabolic engineering to elucidate cancer metabolism

The metabolic engineer's toolbox, comprising stable isotope tracers, flux estimation and analysis, pathway identification, and pathway kinetics and regulation, among other techniques, has long been used to elucidate and quantify pathways primarily in the context of engineering microbes for producing small molecules of interest. Recently, these tools are increasingly finding use in cancer biology due to their unparalleled capacity for quantifying intracellular metabolism of mammalian cells. Here, we review basic concepts that are used to derive useful insights about the metabolism of tumor cells, along with a number of illustrative examples highlighting the fundamental contributions of these methods to elucidating cancer cell metabolism. This area presents unique opportunities for metabolic engineering to expand its portfolio of applications into the realm of cancer biology and help develop new cancer therapies based on a new class of metabolically derived targets.

Abstract 1001: Reductive tricarboxylic acid metabolism of glutamine mediates lipogenesis under hypoxia

Abstract Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and adenosine triphosphate citrate lyase (ACL) in the cytosol. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle (TCA) and fatty acid synthesis. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis, the regulation and utilization of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells employ reductive metabolism of alpha-ketoglutarate (aKG) to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase 1 (IDH1) dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived aKG for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau (VHL) tumor suppressor protein preferentially utilize reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon utilization in order to produce AcCoA and support lipid synthesis in mammalian cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1001. doi:1538-7445.AM2012-1001

Growth, productivity and protein glycosylation in a CHO EpoFc producer cell line adapted to glutamine-free growth

A primary objective of cell line development and process optimisation in animal cell culture is the improvement of culture performance as indicated by desirable properties such as high cell concentration, viability, productivity and product quality. The inefficient energy metabolism of mammalian cells in culture is still a major limiting factor for improvements in process performance. It results in high uptake rates of glucose and glutamine and the concomitant accumulation of waste products which in turn limits final cell concentrations and growth. To avoid these negative side effects, a CHO host cell line was established recently which is able to grow in completely glutamine free medium (Hernandez Bort et al., 2010). To determine the influence of this adaptation on productivity and product quality, the same procedure was repeated with a recombinant CHO cell line producing an erythropoietin-Fc fusion protein (CHO-EpoFc) for this publication. After adaptation to higher cell densities and glutamine free medium, culture performance was monitored in batch bioprocesses and revealed comparable growth properties and EpoFc product formation in both cell lines. The level of reactive oxygen species was elevated in the adapted cells, reflecting a higher level of oxidative stress, however, at the same time the level of the oxido-protective glutathione was also higher, so that cells seem adequately protected against cellular damage. Analysis of nucleotides and nucleotide sugars revealed elevated UDP-sugars in cells grown in the absence of glutamine. Furthermore, the antennarity of N-glycans was moderately higher on the Epo part of the protein produced by the adapted cell line compared to the parental cell line. Except for this, the glycosylation, with respect to site occupancy, degree of sialylation and glycoform structure, was highly comparable, both for the Epo and the Fc part of the protein.

Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia

Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty-acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and ATP citrate lyase in the cytosol. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near-stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle and fatty-acid synthesis. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis, the regulation and use of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells use reductive metabolism of α-ketoglutarate to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase-1 (IDH1)-dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived α-ketoglutarate for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau tumour suppressor protein preferentially use reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon use to produce AcCoA and support lipid synthesis in mammalian cells.

Subcellular signaling in the endothelium: cyclic nucleotides take their place

When lecturing on the topic of cellular signaling I have had occasion to ask the class for examples of cellular processes NOT impacted by cyclic AMP (cAMP) and am struck by how few examples exist. Indeed, studies spanning the past 60 years have detailed how this ubiquitous second messenger impacts virtually all cellular processes, including intermediary metabolism, contractility, motility, proliferation, and gene expression in most mammalian cells. Since the hydrophobic cAMP could in principle diffuse rapidly throughout the cell once formed, the remarkable spatial and temporal specificity of its numerous actions in cells is truly impressive. Herein I introduce the main players involved in coordinating actions of cAMP in vascular endothelial cells (VECs), and focus on the increasing awareness of the dominant role that cyclic nucleotide phosphodiesterases (PDEs), the sole cellular enzymes capable of hydrolytically inactivating cAMP, play in fostering this specificity.

Itaconic Acid Is a Mammalian Metabolite Induced during Macrophage Activation

Itaconic acid (ITA), or methylenesuccinic acid, is not generally classified as a mammalian metabolite. Using NMR-based metabolomics and (13)C-labeling, we have detected ITA in both macrophage-like VM-M3 and RAW 264.7 tumor cell lines as well as stimulated and unstimulated primary murine macrophages. Macrophage activation by addition of lipopolysaccharide and IFN-γ markedly increased ITA production and secretion. Crude cell extracts synthesize ITA via decarboxylation of cis-aconitate, indicative of a novel mammalian cis-aconitic decarboxylase activity. Our results highlight a previously unidentified biosynthetic pathway related to TCA cycle metabolism in mammalian cells and a novel metabolite that likely plays a role in macrophage-based immune response.

Muscle glycogen resynthesis, signalling and metabolic responses following acute exercise in exercise-trained pigs carrying the PRKAG3 mutation

Hampshire pigs carrying the PRKAG3 mutation in the AMP-activated protein kinase (AMPK) γ3 subunit exhibit excessive skeletal muscle glycogen storage and an altered glycogen synthesis signalling response following exercise. AMPK plays an important role as a regulator of carbohydrate and fat metabolism in mammalian cells. Exercise-trained muscles are repeatedly exposed to glycogen degradation and resynthesis, to which the signalling pathways adapt. The aim of this study was to examine the effect of acute exercise on glycogen synthesis signalling pathways, and the levels of insulin and other substrates in blood in exercise-trained pigs with and without the PRKAG3 mutation. After 5 weeks of training, pigs performed two standardized treadmill exercise tests, and skeletal muscle biopsies were obtained immediately after exercise and 3 h postexercise in the first test, and 6 h postexercise in the second test. The PRKAG3 mutation carriers had higher glycogen storage, and resynthesis of glycogen was faster after 3 h but not after 6 h of recovery. Alterations in the concentrations of insulin, glucose, lactate and free fatty acids after exercise did not differ between the genotypes. The carriers showed a lower expression of AMPK and increased phosphorylation of Akt Ser(473) after exercise, compared with non-carriers. Acute exercise stimulated the phosphorylation of AS160 in both genotypes, and the phosphorylation of GSK3α Ser(21) and ACC Ser(79) in the non-carriers. In conclusion, exercise-trained pigs carrying the PRKAG3 mutation show an altered Akt and AMPK signalling response to acute exercise, indicating that glucose metabolism is associated with faster resynthesis of muscle glycogen in this group.

Molecular Crowding Defines a Common Origin for the Warburg Effect in Proliferating Cells and the Lactate Threshold in Muscle Physiology

Aerobic glycolysis is a seemingly wasteful mode of ATP production that is seen both in rapidly proliferating mammalian cells and highly active contracting muscles, but whether there is a common origin for its presence in these widely different systems is unknown. To study this issue, here we develop a model of human central metabolism that incorporates a solvent capacity constraint of metabolic enzymes and mitochondria, accounting for their occupied volume densities, while assuming glucose and/or fatty acid utilization. The model demonstrates that activation of aerobic glycolysis is favored above a threshold metabolic rate in both rapidly proliferating cells and heavily contracting muscles, because it provides higher ATP yield per volume density than mitochondrial oxidative phosphorylation. In the case of muscle physiology, the model also predicts that before the lactate switch, fatty acid oxidation increases, reaches a maximum, and then decreases to zero with concomitant increase in glucose utilization, in agreement with the empirical evidence. These results are further corroborated by a larger scale model, including biosynthesis of major cell biomass components. The larger scale model also predicts that in proliferating cells the lactate switch is accompanied by activation of glutaminolysis, another distinctive feature of the Warburg effect. In conclusion, intracellular molecular crowding is a fundamental constraint for cell metabolism in both rapidly proliferating- and non-proliferating cells with high metabolic demand. Addition of this constraint to metabolic flux balance models can explain several observations of mammalian cell metabolism under steady state conditions.

Molecular crowding defines a common origin for the Warburg effect in proliferating cells and the lactate threshold in muscle physiology

Aerobic glycolysis is a seemingly wasteful mode of ATP production that is seen both in rapidly proliferating mammalian cells and highly active contracting muscles, but whether there is a common origin for its presence in these widely different systems is unknown. To study this issue, here we develop a model of human central metabolism that incorporates a solvent capacity constraint of metabolic enzymes and mitochondria, accounting for their occupied volume densities, while assuming glucose and/or fatty acid utilization. The model demonstrates that activation of aerobic glycolysis is favored above a threshold metabolic rate in both rapidly proliferating cells and heavily contracting muscles, because it provides higher ATP yield per volume density than mitochondrial oxidative phosphorylation. In the case of muscle physiology, the model also predicts that before the lactate switch, fatty acid oxidation increases, reaches a maximum, and then decreases to zero with concomitant increase in glucose utilization, in agreement with the empirical evidence. These results are further corroborated by a larger scale model, including biosynthesis of major cell biomass components. The larger scale model also predicts that in proliferating cells the lactate switch is accompanied by activation of glutaminolysis, another distinctive feature of the Warburg effect. In conclusion, intracellular molecular crowding is a fundamental constraint for cell metabolism in both rapidly proliferating- and non-proliferating cells with high metabolic demand. Addition of this constraint to metabolic flux balance models can explain several observations of mammalian cell metabolism under steady state conditions.

Molecular crowding defines a common origin for the Warburg effect in proliferating cells and the lactate threshold in muscle physiology

AbstractAerobic glycolysis is a seemingly wasteful mode of ATP production that is seen both in rapidly proliferating mammalian cells and highly active contracting muscles, but whether there is a common origin for its presence in these widely different systems is unknown. To study this issue, here we develop a model of human central metabolism that incorporates a solvent capacity constraint of metabolic enzymes and mitochondria, accounting for their occupied volume densities, while assuming glucose and/or fatty acid utilization. The model demonstrates that activation of aerobic glycolysis is favored above a threshold metabolic rate in both rapidly proliferating cells and heavily contracting muscles, because it provides higher ATP yield per volume density than mitochondrial oxidative phosphorylation. In the case of muscle physiology, the model also predicts that before the lactate switch, fatty acid oxidation increases, reaches a maximum, and then decreases to zero with concomitant increase in glucose utilization, in agreement with the empirical evidence. These results are further corroborated by a larger scale model, including biosynthesis of major cell biomass components. The larger scale model also predicts that in proliferating cells the lactate switch is accompanied by activation of glutaminolysis, another distinctive feature of the Warburg effect. In conclusion, intracellular molecular crowding is a fundamental constraint for cell metabolism in both rapidly proliferating- and non-proliferating cells with high metabolic demand. Addition of this constraint to metabolic flux balance models can explain several observations of mammalian cell metabolism under steady state conditions.

Virtual screening, identification and experimental testing of novel inhibitors of PBEF1/Visfatin/NMPRTase for glioma therapy.

BackgroundPre-B-cell colony enhancing factor 1 gene (PBEF1) encodes nicotinamide phosphoribosyltransferase (NMPRTase), which catalyses the rate limiting step in the salvage pathway of NAD+ metabolism in mammalian cells. PBEF1 transcript and protein levels have been shown to be elevated in glioblastoma and a chemical inhibitor of NMPRTase has been shown to specifically inhibit cancer cells.MethodsVirtual screening using docking was used to screen a library of more than 13,000 chemical compounds. A shortlisted set of compounds were tested for their inhibition activity in vitro by an NMPRTase enzyme assay. Further, the ability of the compounds to inhibit glioma cell proliferation was carried out.ResultsVirtual screening resulted in short listing of 34 possible ligands, of which six were tested experimentally, using the NMPRTase enzyme inhibition assay and further with the glioma cell viability assays. Of these, two compounds were found to be significantly efficacious in inhibiting the conversion of nicotinamide to NAD+, and out of which, one compound, 3-amino-2-benzyl-7-nitro-4-(2-quinolyl-)-1,2-dihydroisoquinolin-1-one, was found to inhibit the growth of a PBEF1 over expressing glioma derived cell line U87 as well.ConclusionsThus, a novel inhibitor has been identified through a structure based drug discovery approach and is further supported by experimental evidence.

The role of iron regulatory proteins in the control of iron metabolism in mammals

The iron regulatory proteins (IRP1 and IRP2) are two cytoplasmic RNA-binding proteins involved in the mechanisms that control iron metabolism in mammalian cells. They modulate the expression of iron-related proteins at a post-transcriptional level by binding to specific iron regulatory elements (IREs) on their mRNAs. IRP-IRE interaction can block protein synthesis or stabilize the mRNA. At low intracellular iron concentration, IRPs bind to the IRE of ferritin or ferroportin mRNAs and block their translation. Direct interactions between IRPs and several IRE motifs stabilize transferrin receptor mRNA. The converse regulation of ferritin and TfR synthesis, being a consequence of the lack of binding of IRPs to IRE, occurs in cells with high iron level. Thus, IRP-mediated regulation rapidly restores the physiological level of iron during its deficiency as well as excess. The role of IRPs in maintaining the intracelluar iron balance has been relatively well characterized in numerous types of mammalian cells. However, the importance of IRPs in the regulation of systemic iron metabolism in mammals, particularly, in signaling between the cells which play major roles in body iron metabolism, such as duodenal enterocytes, reticuloendothelial macrophages, hepatocytes, and bone marrow precursors of red blood cells, is only beginning to be investigated. Several studies have shown that IRP2 is a predominant regulator of iron homeostasis in mice housed under standard conditions, thus limiting the impact of IRP1 on this metabolic pathway. Although IRP1-deficient mice do not display a strong pathological phenotype, a deletion of both IRPs is embryonic lethal. In addition, in vitro and in vivo studies have reported that nitric oxide (NO) and hydrogen peroxide (H2O2), which are produced during inflammation, are potent IRP1 regulators that mediate the disassembly of Fe-S cluster of IRP1. There is also an increasing evidence that NO and superoxide anion (O2 ) may induce a strong down-regulation of IRP1 at the protein level and thus have an impact on the binding of IRP1 to IREs. All these data suggest a predominant role of IRP1 in the regulation of iron homeostasis under specific physiopathological conditions.