Comparative Metabolomics Reveals Endogenous Ligands of DAF-12, a Nuclear Hormone Receptor, Regulating C. elegans Development and Lifespan

Abstract

SUMMARY Small-molecule ligands of nuclear hormone receptors (NHRs) govern the transcriptional regulation of metazoan development, cell differentiation, and metabolism. However, the physiological ligands of many NHRs remain poorly characterized primarily due to lack of robust analytical techniques. Using comparative metabolomics, we identified endogenous steroids that act as ligands of the C. elegans NHR, DAF-12, a vitamin-D and liver-X receptor homolog regulating larval development, fat metabolism, and lifespan. The identified molecules feature unexpected chemical modifications and include only one of two DAF-12 ligands reported earlier, necessitating a revision of previously proposed ligand biosynthetic pathways. We further show that ligand profiles are regulated by a complex enzymatic network including the Rieske oxygenase DAF-36, the short-chain dehydrogenase DHS-16, and the hydroxysteroid dehydrogenase, HSD-1. Our results demonstrate the advantages of comparative metabolomics over traditional candidate-based approaches and provide a blueprint for the identification of ligands for other C. elegans and mammalian NHRs.

Steroid hormone signalling links reproduction to lifespan in dietary-restricted Caenorhabditis elegans

“…We found that these genes were dispensable for DR-induced lifespan extension (Supplementary Fig. 5 and Supplementary Table 1), consistent with the notion proposed by others that DAs can be produced through an as yet uncharacterized alternative pathway 12,13 . Finally, we asked whether overproduction of DAs could enhance the response to bacterial deprivation.…”

“…First, NHR-8 could be activated by another form of DA. This is supported by a recent report that a variety of DAs can bind with different affinities and activate DAF-12 in vivo 13 . Second, NHR-8 activation could require binding with co-activators that are present in the worm, but not in HEK293 cells.…”

Integration of carbohydrate metabolism and redox state controls dauer larva formation in Caenorhabditis elegans

“…N2 and daf-16(mu86) were grown on cholesterol or lophenolcontaining plates to obtain L3 larvae or dauer/dauer-like larvae, respectively 25 . Total RNA was isolated using the PureLink RNA Mini Kit (Ambion) and equal amounts of all samples were reverse transcribed with SuperScript III (Invitrogen) using oligo(dT) [12][13][14][15][16][17][18] primers. cDNA quality was tested using standard PCR.…”

“…Three signalling pathways, namely the transforming growth factor (TGF)-b-like (defined by the TGF-b homologue DAF-7), insulin-like (defined by the insulin receptor homologue DAF-2) and cyclic GMP pathways 1 , converge on the activity of a nuclear hormone receptor/ transcription factor, DAF-12 (refs 10,11). DAF-12 binds sterolderived, bile acid-like molecules known as dafachronic acids (DAs) 12,13 . The last, crucial reaction in DA biosynthesis is catalysed by cytochrome P450 (CYP450) DAF-9, which produces a carboxyl group at the C-26 methyl residue of the precursor in three oxidation steps.…”

The mysterious relationship between reproduction and longevity

“…Second, it is possible that NHR-8 is activated by a currently unidentified dafachronic acid metabolite. A recent study demonstrated that several forms of dafachronic acid can activate DAF-12 25 , and this may also be true for NHR-8. Finally, NHR-8 may act downstream of dafachronic acid in an indirect manner.…”