Metabotyping of the <i>C. elegans sir-2.1</i> Mutant Using <i>in Vivo</i> Labeling and ¹³C‑Heteronuclear Multidimensional NMR Metabolomics

The roles of <i>sir-2.1</i> in <i>C. elegans</i> lifespan extension have been subjects of recent public and academic debates. We applied an efficient workflow for <i>in vivo</i> ¹³C-labeling of <i>C. elegans</i> and ¹³C-heteronuclear NMR metabolomics to characterizing the metabolic phenotypes of the <i>sir-2.1</i> mutant. Our method delivered sensitivity 2 orders of magnitude higher than that of the unlabeled approach, enabling 2D and 3D NMR experiments. Multivariate analysis of the NMR data showed distinct metabolic profiles of the mutant, represented by increases in glycolysis, nitrogen catabolism, and initial lipolysis. The metabolomic analysis defined the <i>sir-2.1</i> mutant metabotype as the decoupling between enhanced catabolic pathways and ATP generation. We also suggest the relationship between the metabotypes, especially the branched chain amino acids, and the roles of <i>sir-2.1</i> in the worm lifespan. Our results should contribute to solidifying the roles of <i>sir-2.1</i>, and the described workflow can be applied to studying many other proteins in metabolic perspectives

Metabotyping of the <i>C. elegans sir-2.1</i> Mutant Using <i>in Vivo</i> Labeling and ¹³C‑Heteronuclear Multidimensional NMR Metabolomics

The roles of <i>sir-2.1</i> in <i>C. elegans</i> lifespan extension have been subjects of recent public and academic debates. We applied an efficient workflow for <i>in vivo</i> ¹³C-labeling of <i>C. elegans</i> and ¹³C-heteronuclear NMR metabolomics to characterizing the metabolic phenotypes of the <i>sir-2.1</i> mutant. Our method delivered sensitivity 2 orders of magnitude higher than that of the unlabeled approach, enabling 2D and 3D NMR experiments. Multivariate analysis of the NMR data showed distinct metabolic profiles of the mutant, represented by increases in glycolysis, nitrogen catabolism, and initial lipolysis. The metabolomic analysis defined the <i>sir-2.1</i> mutant metabotype as the decoupling between enhanced catabolic pathways and ATP generation. We also suggest the relationship between the metabotypes, especially the branched chain amino acids, and the roles of <i>sir-2.1</i> in the worm lifespan. Our results should contribute to solidifying the roles of <i>sir-2.1</i>, and the described workflow can be applied to studying many other proteins in metabolic perspectives

A Highly Facile and Specific Assay for Cancer-Causing Isocitrate Dehydrogenase Mutant Using ¹³C₄‑Labeled α‑Ketoglutarate and Heteronuclear NMR

Isocitrate dehydrogenase mutations with neomorphic activity of converting α-ketoglutarate to 2-hydroxyglutarate have been found in many types of cancers. We report an NMR-based assay specific for the mutant using ¹³C₄-labeled α-ketoglutarate. It can be done in a complex mixture without extraction, give time-dependent absolute quantitation, and be applied to enzyme inhibition studies. Its merits over conventional assays should facilitate inhibitor developments for a new class of target-oriented anticancer agents

2–hydroxyglutarate (2HG) levels in the brain samples as measured by <i>ex vivo</i> liquid chromatography–mass spectrometry (LC–MS).

<p>The brain samples were collected from the tumor regions (F98 IDH1/2–WT, IDH1–R132H, and IDH2–R172K) and the contralateral, normal regions (CN VOI). The relative intensity of 2HG in Group A (n = 12) and Group B (n = 7) ranged 0.06x10⁵~0.15x10⁵ and 3.14x10⁵~5.47x10⁵, respectively. Those 6 samples with intermediate 2HG levels (3 from IDH1–R132H and 3 from IDH2–R172K) were excluded in the final data analysis, and then the Group A and Group B were treated as 2HG–absent and 2HG–present, respectively.</p

Establishment of IDH1–wild type (WT), IDH1–mutant (–R132H), IDH2–WT, and IDH2–mutant (–R172K) overexpressing F98 cell lines.

<p>(A) Construction map of IDH1/2–WT or –R132H/–R172K lentiviral vector. (B) GFP expressions in Mock, IDH1/2–WT or –R132H/–R172K vector transduced F98 cells by using Fluorescence–activated cell sorting (FACS). (C) GFP–tagged gene expressions of the F98 cells confirmed by fluorescent microscopic images. (D) Immunoblot analysis where IDH1–R132H or IDH2–R172K specific antibodies were detected only in the mutated epitopes of F98 IDH1–R132H or IDH2–R172K, respectively.</p

Representative 1H–MRS spectra.

<p>(A and C) A contralateral normal brain region (CN VOI). (B and D) A brain tumor region (tumor VOI) with F98 IDH2–R172K glioma. All spectra were post–processed with voxel–specifically obtained spectral baselines by using either MRUI (A and B) or LCModel (C and D). The resulting residual of fit and the 2HG spectral components are also shown, which were denoted by dashed lines in ~1.8–2.3 ppm. (2HG: 2–hydroxyglutarate, GABA: gamma–aminobutylic acid, Gln: glutamine, Glu: glutamate, Lac: lactate, mI: myo–inositol, NAA: N–acetylaspartate, NAAG: N–acetylaspartylglutamate, Tau: taurine, tCho: total choline, tCr: total creatine, tNAA: total N–acetylaspartate).</p

The results of 2HG detection by 1H–MRS.

<p>T_n, true-negative; T_p, true-positive; F_n, false-negative; F_p, false-positive; U, uncertain case (CRLB >20%); n/a, not available;</p