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

Abstract

The roles of sir-2.1 in C. elegans lifespan extension have been subjects of recent public and academic debates. We applied an efficient workflow for in vivo(13)C-labeling of C. elegans and (13)C-heteronuclear NMR metabolomics to characterizing the metabolic phenotypes of the sir-2.1 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 sir-2.1 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 sir-2.1 in the worm lifespan. Our results should contribute to solidifying the roles of sir-2.1, and the described workflow can be applied to studying many other proteins in metabolic perspectives.

13C NMR Metabolomics: Applications at Natural Abundance

“…Because the energy of an NMR resonance transition is proportional to γ 3 , 13 C has an intrinsic sensitivity decrease of nearly 64 (4 3 ) when compared to 1 H. Though solutions such as isotopic labeling exist, 1−5 they can be expensive and are often restricted to targeted metabolomics. Untargeted studies with 13 C labeling are especially challenging for organisms that cannot be cultured.…”

13C NMR Metabolomics: INADEQUATE Network Analysis

“…25,31 Two previous studies used 1 H-detected 1D, 2D TOCSY, 25 2D 13 C-HSQC, and 3D 13 C-HSQC-TOCSY 31 to identify a total of 35 metabolites in the exometabolome. 25,31 Using INETA with the parameters defined in Table 1, we found 11 of the 35 metabolites from previous studies. What about the other 24?…”

“…al 25 and An et. al 31 reported 35 compounds, 11 of which were also found using INETA with the parameters set in Table 1. Targeted manual analysis of the 1D 13 C and INADEQAUTE data yielded an additional 12 metabolites.…”

Multidimensional Approaches to NMR-Based Metabolomics

“…On the other hand, HSQC spectra of individual compounds represent useful fingerprints, providing the number of C-H spin pairs of the molecule together with the 13 C and 1 H chemical shifts, which report on the nature of the chemical groups. 323334 …”