Following their previously published findings (reviewed here on Accelerating Proteomics), Depuydt et al. have now turned their attention to harvesting more data from the proteomic profile of long-lived daf-2 mutant Caenorhabditis elegans worms.1 These worms, which are characterized by disruption of the insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) pathway, live longer than wild-type C. elegans even when nutritionally replete. In addition to prolongation of lifespan, the worms also show increased innate immunity, resistance to stress, and metabolic changes.
In their 2013 study, Depuydt and co-authors found that muscle function and muscle mass itself were maintained in the mutant worms, similar to that seen under experimental hypometabolism induced by dietary restriction. Proteomic profiling showed changes in proteins involved in muscle synthesis and preservation. Other studies confirmed that, proteomically, daf-2 worms showed alterations in carbohydrate metabolism as a result of their attenuated IIS pathway signaling.
Because the longevity of daf-2 C. elegans worms is closely associated with a disrupted IIS pathway, Depuydt et al. wondered if there were other metabolic changes that could be predicted from proteomic profiling. The team used data from their initial accurate mass tagging liquid chromatography–mass spectrometric (AMT LC-MS) analysis, which was conducted using a constant-pressure capillary high-performance liquid chromatography (HPLC) system coupled online with LTQ Orbitrap hybrid ion trap-Orbitrap mass spectrometer technology (Thermo Scientific).
Comparing proteomic profiles from the daf-2 worms with reference strains, the researchers used a variety of software tools to examine changes in protein abundance and relate them to specific cellular pathways. These included the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis tool used alongside the UniProt Knowledgebase to form a C. elegans metabolic network and, for gene set enrichment analysis (GSEA), use of GSEA-P software to determine statistical difference in metabolic pathways.
Depuydt et al. found that functional analysis did indeed show changes at the proteome level, with functional analysis clustering revealing enrichment of proteins involved in metabolism in the long-lived daf-2 mutants. They found increased expression of enzymes involved in gluconeogenesis, glycolysis and glycogenolysis, along with those involved in the citric acid cycle. Furthermore, proteins involved in propionate and amino acid metabolism were also elevated, as were those found as components of the electron transport chain.
The research team examined the worms anatomically, using transmission electron microscopy and Oil-Red-O staining for fat, to see if physical alterations in body composition paralleled the proteome changes. Compared to the reference strain, the daf-2 mutants laid down more fat during early stages of life but then utilized it more slowly. Considered in conjunction with enzyme studies, Depuydt and co-authors conclude that these worms rely more heavily on their internal fat stores for energy, controlling substrate utilization more strictly than do the reference strains.
Overall, do the authors think these changes contribute to the long lifespan of these C. elegans worms? Although they note some seemingly contradictory results with elevated protein levels in pathways reported by others as attenuated in activity, Depuydt et al. conclude that their results show that the daf-2 mutants have developed efficient ways of using their energy stores, stretching out internal resources for a longer life while yet maintaining the ability to respond to their environment. The researchers suggest that data from their experiments will benefit further research into insulin-related disease in man, as well as research on aging and longevity.
1. Depuydt, G.J., et al. (2014) “LC-MS Proteomics Analysis of the Insulin/IGF-1 Deficient C. elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism,” Journal of Proteome Research, 13 (pp. 1938–56), doi: 10.1021/pr401081b.
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