No Significant Increase in the Δ4- and Δ7-Dafachronic Acid Concentration in the Long-Lived glp-1 Mutant, nor in the Mutants Defective in Dauer Formation

Abstract

The steroid hormone dafachronic acid (DA) regulates dauer formation and lifespan in Caenorhabditis elegans by binding to the nuclear receptor DAF-12. However, little is known about how DA concentrations change under various physiologic conditions and about how DA/DAF-12 signaling interacts with other signaling pathways that also regulate dauer formation and lifespan. Using a sensitive bioanalytical method, we quantified the endogenous DA concentrations in a long-lived germline-less glp-1 mutant and in the Dauer formation-defective (Daf-d) mutants daf-12, daf-16, daf-5, and daf-3. We found that the DA concentration in the glp-1 mutant was similar to that in the wild type (WT). This result is contrary to the long-held belief that germline loss-induced longevity involves increased DA production and suggests instead that this type of longevity involves an enhanced response to DA. We also found evidence suggesting that increased DA sensitivity underlies lifespan extension triggered by exogenous DA. At the L2/L3 stage, the DA concentration in a daf-12 null mutant decreased to 22% of the WT level. This finding is consistent with the previously proposed positive feedback regulation between DAF-12 and DA production. Surprisingly, the DA concentrations in the daf-16, daf-5, and daf-3 mutants were only 19-34% of the WT level at the L2/L3 stage, slightly greater than those in the Dauer formation-constitutive (Daf-c) mutants at the pre-dauer stage (4-15% of the WT L2 control). Our experimental evidence suggested that the positive feedback between DA and DAF-12 was partially induced in the three Daf-d mutants.

Keywords: C. elegans dauer; daf-12; dafachronic acid; glp-1; steroid hormone.

Figure 1

Endogenous dafachronic acid (DA) concentrations in the wild type (WT), gon-2(q388), and glp-1(e2141) day-1 adults cultured at 20° or 25°. The DA concentrations were averaged over three independent experiments and normalized to level of the WT samples cultured at 20°. The absolute quantitation values are listed in Table S2. Error bars indicate the SD. Student’s t-tests were used for statistical analysis. n.s., not significant (P > 0.05).

Figure 2

Exogenous dafachronic acid (DA) extended the lifespan of wild type (WT) worms at elevated temperatures in a daf-12-, daf-16-, and hsf-1-dependent manner. (A−C) Exogenous DA supplemented only during adulthood extended the lifespan of WT worms cultured at 25° (B) and 28° (C), but not at 20° (A). (D) WT day-1 adults exhibited weak or strong nuclear accumulation of DAF-16::GFP (indicated by arrows) 12 hr after a temperature shift from 20° to 25° or 28°. (E−J) The lifespan extension effect of DA at 25° and 28° required daf-12 (E, H), daf-16 (F, I), and hsf-1 (G, J). Δ4-DA was not tested in the lifespan assays at 28° nor in the lifespan assay of the hsf-1 mutant at 25°.

Figure 3

Dafachronic acid (DA) concentrations in the Daf-d mutants. (A) Relative DA levels in the Dauer formation-defective Daf-d mutant larvae cultured at 25°, expressed as averages ± SDs (n = 3). The values are listed in Table S5. Student’s t-tests were used for statistical analysis. ***P < 0.001, **P < 0.01, *P < 0.05. (B) mRNA transcripts of daf-9, but not daf-36 or dhs-16, were significantly reduced in the daf-12−null mutant (P = 0.02 for daf-9 mRNA). Results of three independent biological replicates are shown. (C) Model of DA regulation in dauer formation.