Abstract
Abnormal nutrient metabolism is a hallmark of aging, and the underlying genetic and nutritional framework is rapidly being uncovered, particularly using C. elegans as a model. However, the direct metabolic consequences of perturbations in life history of C. elegans remain to be clarified. Based on recent advances in the metabolomics field, we optimized and validated a sensitive mass spectrometry (MS) platform for identification of major metabolite classes in worms and applied it to study age and diet related changes. Using this platform that allowed detection of over 600 metabolites in a sample of 2500 worms, we observed marked changes in fatty acids, amino acids and phospholipids during worm life history, which were independent from the germ-line. Worms underwent a striking shift in lipid metabolism after early adulthood that was at least partly controlled by the metabolic regulator AAK-2/AMPK. Most amino acids peaked during development, except aspartic acid and glycine, which accumulated in aged worms. Dietary intervention also influenced worm metabolite profiles and the regulation was highly specific depending on the metabolite class. Altogether, these MS-based methods are powerful tools to perform worm metabolomics for aging and metabolism-oriented studies.
Highlights
Aging was long considered a passive process that could not be reversed
For fatty acid (FA), the limit of detection (LOD) was between 0.006–0.01 nmol/ mg protein, and the limit of quantification (LOQ) was 0.02–0.03 nmol/mg protein, allowing quantification of 35 FA species; for amino acid (AA), the LOD was between 0.1–2 nmol/mg protein and the LOQ was 0.3–4 nmol/mg protein
Taking into consideration both the LOD/LOQ and the ease of preparing worm samples, we chose to continue our validation with 150 μg of protein for FA and 50 μg for AA, an amount that corresponds to approximately 500 worms and allows extraction of both metabolite classes from the same lysate
Summary
Aging was long considered a passive process that could not be reversed. Over the recent decades, prolonged life expectancy in economically developed countries is proposed as a positive outcome of progress in medicine and modern lifestyle. Several studies in the nematode Caenorhabditis elegans showed that developmental rate, reproduction and lifespan were influenced when worms were fed with different bacterial strains[7,8,9]. Together, these studies accentuate the central role of metabolism in controlling animal lifespan. Genetic intervention studies demonstrated the involvement of metabolic pathways, e.g. proline metabolism, in the regulation of aging in the long-lived daf-2 (insulin receptor) mutant worms, as well as in worms exposed to different bacterial strains as dietary sources[8, 12]. Our data highlight how application of metabolomics is a powerful tool to uncover mechanisms that underlie the link between metabolic changes and aging in C. elegans
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