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

ABSTRACT: 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-(13)C]-glucose and [U-(13)C,(15)N]-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 (15)N NMR. The incorporation of (13)C 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.

SUBMITTER: Fan TW

PROVIDER: S-EPMC4486296 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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

Fan Teresa W-M TW Tan Jinlian J McKinney Martin M MM Lane Andrew N AN

Metabolomics : Official journal of the Metabolomic Society 20120601 3

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 a ...[more]

PMID: 26146495

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Project description:Amino acids have crucial roles in central metabolism, both anabolic and catabolic. To elucidate these roles, steady-state concentrations of amino acids alone are insufficient, as each amino acid participates in multiple pathways and functions in a complex network, which can also be compartmentalized. Stable Isotope-Resolved Metabolomics (SIRM) is an approach that uses atom-resolved tracking of metabolites through biochemical transformations in cells, tissues, or whole organisms. Using different elemental stable isotopes to label multiple metabolite precursors makes it possible to resolve simultaneously the utilization of these precursors in a single experiment. Conversely, a single precursor labeled with two (or more) different elemental isotopes can trace the allocation of e.g. C and N atoms through the network. Such dual-label experiments however challenge the resolution of conventional mass spectrometers, which must distinguish the neutron mass differences among different elemental isotopes. This requires ultrahigh resolution Fourier transform mass spectrometry (UHR-FTMS). When combined with direct infusion nano-electrospray ion source (nano-ESI), UHR-FTMS can provide rapid, global, and quantitative analysis of all possible mass isotopologues of metabolites. Unfortunately, very low mass polar metabolites such as amino acids can be difficult to analyze by current models of UHR-FTMS, plus the high salt content present in typical cell or tissue polar extracts may cause unacceptable ion suppression for sources such as nano-ESI. Here we describe a modified method of ethyl chloroformate (ECF) derivatization of amino acids to enable rapid quantitative analysis of stable isotope labeled amino acids using nano-ESI UHR-FTMS. This method showed excellent linearity with quantifiable limits in the low nanomolar range represented in microgram quantities of biological specimens, which results in extracts with total analyte abundances in the low to sub-femtomole range. We have applied this method to profile amino acids and their labeling patterns in 13C and 2H doubly labeled PC9 cell extracts, cancerous and non-cancerous tissue extracts from a lung cancer patient and their protein hydrolysates as well as plasma extracts from mice fed with a liquid diet containing 13C6-glucose (Glc). The multi-element isotopologue distributions provided key insights into amino acid metabolism and intracellular pools in human lung cancer tissues in high detail. The 13C labeling of Asp and Glu revealed de novo synthesis of these amino acids from 13C6-Glc via the Krebs cycle, specifically the elevated level of 13C3-labeled Asp and Glu in cancerous versus non-cancerous lung tissues was consistent with enhanced pyruvate carboxylation. In addition, tracking the fate of double tracers, (13C6-Glc + 2H2-Gly or 13C6-Glc + 2H3-Ser) in PC9 cells clearly resolved pools of Ser and Gly synthesized de novo from 13C6-Glc (13C3-Ser and 13C2-Gly) versus Ser and Gly derived from external sources (2H3-Ser, 2H2-Gly). Moreover the complex 2H labeling patterns of the latter were results of Ser and Gly exchange through active Ser-Gly one-carbon metabolic pathway in PC9 cells.

Project description: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.

Dataset Information

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

Publications

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

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