Stable Isotope-resolved Metabolomics Research Articles

Antagonistic effects of MYC and hypoxia in channeling glucose and glutamine into de novo nucleotide biosynthesis

Background Cell proliferation requires up regulation of nucleotide biosynthesis for making new DNA and RNA to support protein bio-synthesis. MYC is a major transcription factor that regulates metabolic processes essential for cell division, and is overexpressed in many cancers. The nutrient sources and integration of the metabolic pathways for nucleotide biosynthesis that enable MYC-dependent cell division are poorly defined. Using Stable Isotope Resolved Metabolomics (SIRM) we have determined the fate of atoms from C6-glucose, C5, N2-glutamine, or H-glycine into nucleotides under varied conditions of MYC expression in the MYC-inducible P493-6 B-lymphocyte [2] and several lung cell lines.

Stable isotope-resolved metabolomic (SIRM) approach: From bench to bedside

Stable isotope-resolved metabolomic (SIRM) approach: From bench to bedside

Chapter Nine - Pulsed Stable Isotope-Resolved Metabolomic Studies of Cancer Cells

Metabolic reprogramming is a key step in oncogenic transformation, and it involves alterations in both bioenergetic and anabolic metabolism. Sustained by these metabolic alterations, malignant cells acquire the ability to re-enter the cell cycle and proliferate. The so-called central carbon metabolism (CCM) is the ultimate source for energy and building blocks enabling cellular growth and proliferation. The time-resolved monitoring of the conversion of stable isotope-labeled metabolites provides profound insights into the metabolic dynamics of malignant cells and enables the tracking of individual carbon routes within the CCM. Specifically, the analysis of isotope incorporation rates within short time frames by means of pulsed stable isotope-resolved metabolomics (pSIRM) can be used to determine the dynamics of glycolysis and glutaminolysis-two metabolic circuitries that are often deregulated in malignant cells. Here, we detail a pSIRM-based method that can be applied to the study of metabolic alteration in cultured cancer cells.

A Computational Framework for High-Throughput Isotopic Natural Abundance Correction of Omics-Level Ultra-High Resolution FT-MS Datasets.

New metabolomics applications of ultra-high resolution and accuracy mass spectrometry can provide thousands of detectable isotopologues, with the number of potentially detectable isotopologues increasing exponentially with the number of stable isotopes used in newer isotope tracing methods like stable isotope-resolved metabolomics (SIRM) experiments. This huge increase in usable data requires software capable of correcting the large number of isotopologue peaks resulting from SIRM experiments in a timely manner. We describe the design of a new algorithm and software system capable of handling these high volumes of data, while including quality control methods for maintaining data quality. We validate this new algorithm against a previous single isotope correction algorithm in a two-step cross-validation. Next, we demonstrate the algorithm and correct for the effects of natural abundance for both 13C and 15N isotopes on a set of raw isotopologue intensities of UDP-N-acetyl-D-glucosamine derived from a 13C/15N-tracing experiment. Finally, we demonstrate the algorithm on a full omics-level dataset.

Fluxomics by NMR Spectroscopy from Cells to Organisms Focusing on Liver

Metabolism represents interconnected networks of metabolite consumption and creation. The field of metabolomics is focused on metabolite concentrations in metabolic networks. Fluxomics quantifies the flux of substrate through each reaction step or a series of reaction steps and is required for energy balance equations of the system. Quantifying metabolic flux of every pathway is unnecessary because there is at least one regulatory enzyme per pathway. Hence, to predict flux for intermediate metabolic pathways, in silico models typically use the total flux of a pathway merged with literature derived steady-state concentration and kinetic values (Vmax and km) as computational conditions. The assumptions of the model can be false or unreliable in the disease state or with xenobiotic exposure. Therefore, the relatively small amount of experimental flux data used in metabolic model must be robust and reliable. Presently, there is no review that lists the multitude of bioenergetics flux values for multiple species, while also highlighting potential errors associated with experimental procedures or data analysis. In this review, the flux values for multiple metabolic pathways in liver of whole animal, perfused liver, and hepatocyte culture studies will be reviewed. Also, the potential pitfalls of experimental stable isotope resolved metabolomics (SIRM) data are described. For animal studies, the main causes of inter-laboratory variance are the effects of anesthesia, adrenergic response during animal handling, variation in tissue sample acquisition and metabolic quenching, and tissue extraction procedures. Solutions to these issues will be discussed. While cell and organ models hope to reduce the complexity of the whole organism to a controlled system of two or more compartments, they do not fully mimic the whole animal inter-organ response and the dynamic concentrations of the metabolite, gases, soluble or insoluble factors in the central compartment. To demonstrate the effects of these factors, studies of the effect of ethanol on hepatocyte, perfused liver and animal studies will be compared, while SIRM studies of human and rat hepatocyte cultures will demonstrate the problem of cell culture in mimicking the normal metabolism.

Glucose-Independent Glutamine Metabolism via TCA Cycling for Proliferation and Survival in B Cells

Because MYC plays a causal role in many human cancers, including those with hypoxic and nutrient-poor tumor microenvironments, we have determined the metabolic responses of a MYC-inducible human Burkitt lymphoma model P493 cell line to aerobic and hypoxic conditions, and to glucose deprivation, using stable isotope-resolved metabolomics. Using [U-(13)C]-glucose as the tracer, both glucose consumption and lactate production were increased by MYC expression and hypoxia. Using [U-(13)C,(15)N]-glutamine as the tracer, glutamine import and metabolism through the TCA cycle persisted under hypoxia, and glutamine contributed significantly to citrate carbons. Under glucose deprivation, glutamine-derived fumarate, malate, and citrate were significantly increased. Their (13)C-labeling patterns demonstrate an alternative energy-generating glutaminolysis pathway involving a glucose-independent TCA cycle. The essential role of glutamine metabolism in cell survival and proliferation under hypoxia and glucose deficiency makes them susceptible to the glutaminase inhibitor BPTES and hence could be targeted for cancer therapy.

Glutamine drives glucose-independent TCA cycle

Methods: We used high-field NMR, in parallel with GC-MS, and direct infusion nanospray FT-ICR-MS as well as stable isotoperesolved metabolomics to trace the fate of 13C and 15N from labeled glucose or glutamine in the P493 human B lymphoma under aerobic, hypoxic glucose-deprived conditions. Levels of specific enzymes, which are involved in glucose and glutamine metabolism, were determined by immunoblotting and LC-MRM-MS and compared with the metabolomic profiles.

High information throughput analysis of nucleotides and their isotopically enriched isotopologues by direct-infusion FTICR-MS

Fourier transform-ion cyclotron resonance-mass spectrometry (FTICR-MS) is capable of acquiring unmatched quality of isotopologue data for stable isotope resolved metabolomics (SIRM). This capability drives the need for a continuous ion introduction for obtaining optimal isotope ratios. Here we report the simultaneous analysis of mono and dinucleotides from crude polar extracts by FTICR-MS by adapting an ion-pairing sample preparation method for LC-MS analysis. This involves a rapid cleanup of extracted nucleotides on pipet tips containing a C(18) stationary phase, which enabled global analysis of nucleotides and their (13)C isotopologues at nanomolar concentrations by direct infusion nanoelectrospray FTICR-MS with 5 minutes of data acquisition. The resolution and mass accuracy enabled computer-assisted unambiguous assignment of most nucleotide species, including all phosphorylated forms of the adenine, guanine, uracil and cytosine nucleotides, NAD(+), NADH, NADP(+), NADPH, cyclic nucleotides, several UDP-hexoses, and all their (13)C isotopologues. The method was applied to a SIRM study on human lung adenocarcinoma A549 cells grown in [U-(13)C] glucose with or without the anti-cancer agent methylseleninic acid. At m/z resolving power of 400,000, (13)C-isotopologues of nucleotides were fully resolved from all other elemental isotopologues, thus allowing their (13)C fractional enrichment to be accurately determined. The method achieves both high sample and high information throughput analysis of nucleotides for metabolic pathway reconstruction in SIRM investigations.

Stable Isotope Resolved Metabolomics Analysis of Ribonucleotide and RNA Metabolism in Human Lung Cancer Cells.

We have developed a simple NMR-based method to determine the turnover of nucleotides and incorporation into RNA by stable isotope resolved metabolomics (SIRM) in A549 lung cancer cells. This method requires no chemical degradation of the nucleotides or chromatography. During cell growth, the free ribonucleotide pool is rapidly replaced by de novo synthesized nucleotides. Using [U-13C]-glucose and [U-13C,15N]-glutamine as tracers, we showed that virtually all of the carbons in the nucleotide riboses were derived from glucose, whereas glutamine was preferentially utilized over glucose for pyrimidine ring biosynthesis, via the synthesis of Asp through the Krebs cycle. Incorporation of the glutamine amido nitrogen into the N3 and N9 positions of the purine rings was also demonstrated by proton-detected 15N NMR. The incorporation of 13C from glucose into total RNA was measured and shown to be a major sink for the nucleotides during cell proliferation. This method was applied to determine the metabolic action of an anti-cancer selenium agent (methylseleninic acid or MSA) on A549 cells. We found that MSA inhibited nucleotide turnover and incorporation into RNA, implicating an important role of nucleotide metabolism in the toxic action of MSA on cancer cells.

A novel deconvolution method for modeling UDP-N-acetyl-D-glucosamine biosynthetic pathways based on 13C mass isotopologue profiles under non-steady-state conditions

BackgroundStable isotope tracing is a powerful technique for following the fate of individual atoms through metabolic pathways. Measuring isotopic enrichment in metabolites provides quantitative insights into the biosynthetic network and enables flux analysis as a function of external perturbations. NMR and mass spectrometry are the techniques of choice for global profiling of stable isotope labeling patterns in cellular metabolites. However, meaningful biochemical interpretation of the labeling data requires both quantitative analysis and complex modeling. Here, we demonstrate a novel approach that involved acquiring and modeling the timecourses of 13C isotopologue data for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) synthesized from [U-13C]-glucose in human prostate cancer LnCaP-LN3 cells. UDP-GlcNAc is an activated building block for protein glycosylation, which is an important regulatory mechanism in the development of many prominent human diseases including cancer and diabetes.ResultsWe utilized a stable isotope resolved metabolomics (SIRM) approach to determine the timecourse of 13C incorporation from [U-13C]-glucose into UDP-GlcNAc in LnCaP-LN3 cells. 13C Positional isotopomers and isotopologues of UDP-GlcNAc were determined by high resolution NMR and Fourier transform-ion cyclotron resonance-mass spectrometry. A novel simulated annealing/genetic algorithm, called 'Genetic Algorithm for Isotopologues in Metabolic Systems' (GAIMS) was developed to find the optimal solutions to a set of simultaneous equations that represent the isotopologue compositions, which is a mixture of isotopomer species. The best model was selected based on information theory. The output comprises the timecourse of the individual labeled species, which was deconvoluted into labeled metabolic units, namely glucose, ribose, acetyl and uracil. The performance of the algorithm was demonstrated by validating the computed fractional 13C enrichment in these subunits against experimental data. The reproducibility and robustness of the deconvolution were verified by replicate experiments, extensive statistical analyses, and cross-validation against NMR data.ConclusionsThis computational approach revealed the relative fluxes through the different biosynthetic pathways of UDP-GlcNAc, which comprises simultaneous sequential and parallel reactions, providing new insight into the regulation of UDP-GlcNAc levels and O-linked protein glycosylation. This is the first such analysis of UDP-GlcNAc dynamics, and the approach is generally applicable to other complex metabolites comprising distinct metabolic subunits, where sufficient numbers of isotopologues can be unambiguously resolved and accurately measured.

Abstract 1271: Human non small cell lung cancer tissue has enhanced expression of pyruvate carboxylase, but not glutaminase

Abstract Altered cellular metabolism in tumors is well established, particularly an increased rate of aerobic glycolysis [1]. However, increased glycolysis alone is insufficient to sustain the enhanced cell growth and anabolic metabolism characteristic of cancer. Another key source of these precursors is the citric acid cycle (CAC). Removal of CAC intermediates for anabolism requires replenishment by other sources; these anaplerotic reactions are vital to cancer development. The two major pathways of anaplerosis are glutaminolyis (deamidation and transamination of glutamine to 2-oxoglutarate) [2] and carboxylation of pyruvate to oxaloacetate (OAA), catalyzed by pyruvate carboxylase (PCB) [2]. We have coupled 13C-labeled glucose tracer with stable isotope resolved metabolomics (SIRM), gene microarray, and western blotting to track the anaplerotic PCB pathway in human lung cancer patients. The resected cancer tissue showed increased flux through the PCB pathway, along with increased transcription of the PCB gene and PCB protein expression compared with the adjacent benign lung tissue [3]. We have extended these findings to 50 paired tissue samples, and have correlated them with histological and clinical data. The protein expression of PCB was on average ten-fold higher in the tumor than in the benign lung tissue (p=0.00001, paired t-test). The normalized protein expression correlated with the fraction of the tumor that was identified as cancer cells, indicating that the increased expression was from the cancer cells rather than other lung cell types. Glutaminase was active in the lung tissue, but its expression was not different from, or lower in the tumors than in the benign lung tissue. These data indicate that in NSCLC, PCB is important in anaplerosis and tumor metabolism. The balance between glycolysis and the choice of different anaplerotic pathways may depend on the tissue types.

Abstract 1251: Anti-cancer mechanisms of se compounds: A stable isotope-resolved metabolomic (SIRM) approach

Abstract Recent rapid advances in analytical tools, especially NMR and mass spectrometry, allow an unprecedented range of metabolites and their transformation pathways to be determined, which transcends classical targeted metabolic studies. We have coupled these tools with stable isotopic tracers [1-3] to investigate the anticancer efficacy and mechanism(s) of different selenium forms using lung adenocarcinoma A549 cells as a model. Selenite (SeO3), methylseleninic acid (MSA) and selenomethionine (SeM) induced distinct changes in steady-state concentrations and 13C labeling patterns of various metabolites including amino & organic acids, nucleotides, antioxidants, as well as phospholipids (PL) and PL metabolites. These changes were determined using a combination of multi-nuclear 2-D NMR, GC-MS, and FT-ICR-MS techniques, which enabled perturbations in multiple metabolic pathways to be simultaneously reconstructed for relating to phenotypic changes. At low µM levels, both SeO3 and MSA inhibited growth, caused cytoskeleton disintegration, and reduced the mitochondrial redox potential in A549 cells. SeM was growth-stimulatory at ≤ 10 µM and inhibited growth but only at 40-fold higher concentration and the extent was much less than MSA or SeO3. These phenotypic differences were related to the early inhibition of glutaminolysis, Krebs cycle, and protein biosynthesis by SeO3, rapid inhibition of pyruvate carboxylation by MSA. SeM had few comparable metabolic effects. Subsequent inhibition of PL biosynthesis was also more substantial in MSA and SeO3 than in SeM-treated cells. The protein and gene expression for key enzymes in these pathways were probed to discern altered metabolic regulations. Thus, the anti-cancer action of MSA differed from that of SeO3 while SeM was not effective against lung cancer cells. The SIRM approach facilitated the resolution of the complex, form-specific anticancer mechanism of Se compounds and is generally applicable to other cancer research problems. 1. Fan, T., et al., Metabolomics-edited transcriptomics analysis of Se anticancer action in human lung cancer cells. Metabolomics Journal, 2005. 1(4): p. 325-339 2. Lane, A.N. and T.W.-M. Fan, Quantification and identification of isotopomer distributions of metabolites in crude cell extracts using 1H TOCSY. Metabolomics, 2007. 3: p. 79-86. 3. Lane, A.N., et al., Isotopomer analysis of lipid biosynthesis by high resolution mass spectrometry and NMR. Analytica Chimica Acta, 2009. 651: p. 201-208. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1251. doi:10.1158/1538-7445.AM2011-1251

ER stress modulates cellular metabolism

Changes in metabolic processes play a critical role in the survival or death of cells subjected to various stresses. In the present study, we have investigated the effects of ER (endoplasmic reticulum) stress on cellular metabolism. A major difficulty in studying metabolic responses to ER stress is that ER stress normally leads to apoptosis and metabolic changes observed in dying cells may be misleading. Therefore we have used IL-3 (interleukin 3)-dependent Bak-/-Bax-/- haemopoietic cells which do not die in the presence of the ER-stress-inducing drug tunicamycin. Tunicamycin-treated Bak-/-Bax-/- cells remain viable, but cease growth, arresting in G1-phase and undergoing autophagy in the absence of apoptosis. In these cells, we used NMR-based SIRM (stable isotope-resolved metabolomics) to determine the metabolic effects of tunicamycin. Glucose was found to be the major carbon source for energy production and anabolic metabolism. Following tunicamycin exposure, glucose uptake and lactate production are greatly reduced. Decreased 13C labelling in several cellular metabolites suggests that mitochondrial function in cells undergoing ER stress is compromised. Consistent with this, mitochondrial membrane potential, oxygen consumption and cellular ATP levels are much lower compared with untreated cells. Importantly, the effects of tunicamycin on cellular metabolic processes may be related to a reduction in cell-surface GLUT1 (glucose transporter 1) levels which, in turn, may reflect decreased Akt signalling. These results suggest that ER stress exerts profound effects on several central metabolic processes which may help to explain cell death arising from ER stress in normal cells.

Stable isotope-resolved metabolomics (SIRM) in cancer research with clinical application to nonsmall cell lung cancer.

Metabolomics provides a readout of the state of metabolism in cells or tissue and their responses to external perturbations. For this reason, the approach has great potential in clinical diagnostics. Clinical metabolomics using stable isotope resolved metabolomics (SIRM) for pathway tracing represents an important new approach to obtaining metabolic parameters in human cancer subjects in situ. Here we provide an overview of the technology development of labeling from cells in culture and mouse models. The high throughput analytical methods NMR and mass spectrometry, especially Fourier transform ion cyclotron resonance, for analyzing the resulting metabolite isotopomers and isotopologues are described with examples of applications in cancer biology. Special technical considerations for clinical applications of metabolomics using stable isotope tracers are described. The whole process from concept to analysis will be exemplified by our on-going study of nonsmall cell lung cancer (NSCLC) metabolomics. This powerful new approach has already provided important new insights into metabolic adaptations in lung cancer cells, including the upregulation of anaplerosis via pyruvate carboxylation in NSCLC.

Stable isotope resolved metabolomics of primary human hepatocytes reveals a stressed phenotype

The development of techniques allowing the culturing of primary mammalian hepatocytes has provided great insights into liver physiology. For most applications, it is desirable for hepatocytes in culture to mimic hepatocytes in vivo. We used stable isotope resolved metabolomics (SIRM) to assess glucose and glutamine utilization in primary rat and human hepatocytes maintained in standard culture media. Primary rat hepatocytes readily metabolized 13C-glucose and 13C-glutamine made evident by 13C incorporation into glycogen, glycolytic end products, and Krebs cycle intermediates and responded to insulin and glucagon appropriately. In contrast, no glucose or glutamine consumption was detected in primary human hepatocytes over 4 h of exposure to high media concentrations of 13C-glucose or 13C-glutamine even in the presence of insulin. Nonetheless, cultured human hepatocytes were metabolically active and viable, as demonstrated by incorporation of media 13C-octanoic acid into Krebs cycle intermediates and ketone bodies. The failure to utilize glucose was not due to inhibition of glucokinase (hexokinase IV) since the human hepatocytes could readily incorporate 13C-glucose into glucuronic acid, as demonstrated by the production of 13C-glucuronide conjugates after addition of acetaminophen to the media. These novel observations support inhibition of phosphofructokinase-1, the other regulatory enzyme in glycolysis. Parts of this phenotype could be reproduced in the rat hepatocytes by replacing insulin with glucagon to the media. We conclude that under standard culture conditions human hepatocytes are in an extreme starved state. We believe this may result from prolonged fasting in the human liver donors combined with exposure to stress hormones such as, epinephrine, glucagon, and cortisol. Efforts should now be exerted to find culture conditions that will reverse this state to achieve more metabolically relevant cultures of human hepatocytes.

Stable isotope resolved metabolomics of lung cancer in a SCID mouse model

We have determined the time course of [U-(13)C]-glucose utilization and transformations in SCID mice via bolus injection of the tracer in the tail vein. Incorporation of (13)C into metabolites extracted from mouse blood plasma and several tissues (lung, heart, brain, liver, kidney, and skeletal muscle) were profiled by NMR and GC-MS, which helped ascertain optimal sampling times for different target tissues. We found that the time for overall optimal (13)C incorporation into tissue was 15-20 min but with substantial differences in (13)C labeling patterns of various organs that reflected their specific metabolism. Using this stable isotope resolved metabolomics (SIRM) approach, we have compared the (13)C metabolite profile of the lungs in the same mouse with or without an orthotopic lung tumor xenograft established from human PC14PE6 lung adenocarcinoma cells. The (13)C metabolite profile shows considerable differences in [U-(13)C]-glucose transformations between the two lung tissues, demonstrating the feasibility of applying SIRM to investigate metabolic networks of human cancer xenograft in the mouse model.

Stable isotope-resolved metabolomic analysis of lithium effects on glial-neuronal metabolism and interactions

Despite the long-established therapeutic efficacy of lithium in the treatment of bipolar disorder (BPD), its molecular mechanism of action remains elusive. Newly developed stable isotope-resolved metabolomics (SIRM) is a powerful approach that can be used to elucidate systematically how lithium impacts glial and neuronal metabolic pathways and activities, leading ultimately to deciphering its molecular mechanism of action. The effect of lithium on the metabolism of three different (13)C-labeled precursors ([U-(13)C]-glucose, (13)C-3-lactate or (13)C-2,3-alanine) was analyzed in cultured rat astrocytes and neurons by nuclear magnetic resonance (NMR) spectroscopy and gas chromatography mass spectrometry (GC-MS). Using [U-(13)C]-glucose, lithium was shown to enhance glycolytic activity and part of the Krebs cycle activity in both astrocytes and neurons, particularly the anaplerotic pyruvate carboxylation (PC). The PC pathway was previously thought to be active in astrocytes but absent in neurons. Lithium also stimulated the extracellular release of (13)C labeled-lactate, -alanine (Ala), -citrate, and -glutamine (Gln) by astrocytes. Interrogation of neuronal pathways using (13)C-3-lactate or (13)C-2,3-Ala as tracers indicated a high capacity of neurons to utilize lactate and Ala in the Krebs cycle, particularly in the production of labeled Asp and Glu via PC and normal cycle activity. Prolonged lithium treatment enhanced lactate metabolism via PC but inhibited lactate oxidation via the normal Krebs cycle in neurons. Such lithium modulation of glycolytic, PC and Krebs cycle activity in astrocytes and neurons as well as release of fuel substrates by astrocytes should help replenish Krebs cycle substrates for Glu synthesis while meeting neuronal demands for energy. Further investigations into the molecular regulation of these metabolic traits should provide new insights into the pathophysiology of mood disorders and early diagnostic markers, as well as new target(s) for effective therapies.

Altered regulation of metabolic pathways in human lung cancer discerned by 13C stable isotope-resolved metabolomics (SIRM)

BackgroundMetabolic perturbations arising from malignant transformation have not been systematically characterized in human lung cancers in situ. Stable isotope resolved metabolomic analysis (SIRM) enables functional analysis of gene dysregulations in lung cancer. To this purpose, metabolic changes were investigated by infusing uniformly labeled 13C-glucose into human lung cancer patients, followed by resection and processing of paired non-cancerous lung and non small cell carcinoma tissues. NMR and GC-MS were used for 13C-isotopomer-based metabolomic analysis of the extracts of tissues and blood plasma.ResultsMany primary metabolites were consistently found at higher levels in lung cancer tissues than their surrounding non-cancerous tissues. 13C-enrichment in lactate, Ala, succinate, Glu, Asp, and citrate was also higher in the tumors, suggesting more active glycolysis and Krebs cycle in the tumor tissues. Particularly notable were the enhanced production of the Asp isotopomer with three 13C-labeled carbons and the buildup of 13C-2,3-Glu isotopomer in lung tumor tissues. This is consistent with the transformations of glucose into Asp or Glu via glycolysis, anaplerotic pyruvate carboxylation (PC), and the Krebs cycle. PC activation in tumor tissues was also shown by an increased level of pyruvate carboxylase mRNA and protein.ConclusionPC activation – revealed here for the first time in human subjects – may be important for replenishing the Krebs cycle intermediates which can be diverted to lipid, protein, and nucleic acid biosynthesis to fulfill the high anabolic demands for growth in lung tumor tissues. We hypothesize that this is an important event in non-small cell lung cancer and possibly in other tumor development.