PDHK-2 and Fat Consumption in C. elegans Survival Figure 1. pdhk-2 mRNA levels under various conditions. Changes in pdhk-2 gene expression were determined by real-time qPCR. (A) Expression levels of pdhk-2 in daf-2(e1370), daf-7(e1372), nhr-49(nr2041), and daf-16(mu86) mutants from well-fed L4 stage worms. The data were normalized to N2 well-fed L4-stage worms. (B) Expression levels of pdhk-2 in N2 worms starved for 2, 6 and 12 hours from the L4 stage, and in dauer and post-dauer stage worms. Dauer worms were obtained after starving for 7 days at 20uC, whereas post-dauer worms were produced by feeding dauer worms for 12-hour at 20uC. The values were normalized to well-fed conditions at the L4 stage worms. Worms maintained under well-fed conditions at the L3 stage were used as controls for the dauer stage group. (C) Expression levels of pdhk-2 in daf-2(e1370) and daf-7(e1372) mutants in the pre-dauer (L1+36 hours) and dauer (L1+60 hours) states at 25uC. The values were normalized to pre-dauer (L1+36 hours) worms. (D) Expression levels of pdhk-2, nhr-49 and daf-16 in N2, daf-16(mu86) and nhr-49(nr2041) worms under 12-hour starvation conditions. In each experiment, mutant worms maintained under well-fed conditions at the L4 stage were used as controls for the starvation group. The values represent means [6SDs] from three independent experiments. *p,0.05, **p,0.01 and ***p,0.001 compared with the control. doi:10.1371/journal.pone.0041755.g001 lipases [14]. Because it appears that the survival of C. elegans dauer larvae during the long-term dauer state heavily depends on the utilization of stored fat, the metabolic regulation of fats (lipolysis, mobilization, and utilization) is a key factor in determining survival rate [14]. Given the substantial induction (,8-fold in starved N2) of pdhk-2 expression in the dauer state, we investigated the potential role of PDHK-2 in the survival of worms in the dauer state using daf-2(e1370);pdhk-2(tm3075) and daf-2(e1370);pdhk- 2(tm3086) double mutants. Our goal was to correlate changes in fat content, if any, with changes in the MLS of these mutants under well-fed conditions and in short-term (day 1) and long-term (day 10) dauer states. First, in the well-fed state, as was previously observed in the pdhk-2 single mutants (Figure S2A), there was no noticeable difference in MLS between daf-2(e1370);pdhk-2(tm3075) and daf- 2(e1370);pdhk-2(tm3086) double mutants (40.162.1 and 44.561.8 days, respectively) and daf-2(e1370) control worms (45.561.3 days) (Figure 3A). Oil Red O staining assays also revealed no significant differences in fat density or TG quantity among the mutant strains tested (Figure 3B and Figure S2C). Moreover, neither dauer formation rates (at 20uC and 25uC) nor dauer recovery rates were different between daf-2(e1370);pdhk-2 double mutants and daf- 2(e1370) worms (data not shown). Second, in the short-term dauer state, there was no significant difference in fat content between daf-2(e1370);pdhk-2 double mutants and daf-2(e1370) control worms measured by Oil Red O staining (fats) and biochemical assays (TG levels) (Figure 3C, E), indicating that PDHK-2 may not be involved in fat metabolism in the early dauer state. However, in long-term dauers, there was generally more fat and fat granules were larger in daf- 2(e1370);pdhk-2(tm3075) and daf-2(e1370);pdhk-2(tm3086) double mutants compared to daf-2(e1370) worms (Figure 3D, E), suggesting a close association between PDHK-2 and fat consump- tion in the late dauer state. Third, measurements of fats remaining in the body of daf- 2(e1370) controls and daf-2(e1370);pdhk-2(tm3075) and daf- 2(e1370);pdhk-2(tm3086) double mutants revealed more residual TGs in the bodies of daf-2(e1370);pdhk-2 double mutants (tm3075, 65.9%; tm3086, 87.1%) compared to daf-2(e1370) worms (54.5%). These results imply that fat consumption is significantly attenuated by a deficiency of PDHK-2 activity. Lastly, we sought to determine how the amount of fat deposits remaining in the body of daf-2(e1370);pdhk-2 double mutants influenced the survival rate of dauer larvae. We found that the survival rate, measured as 50% survival population (S50), was higher for daf-2(e1370);pdhk-2(tm3075) (15.461.88 days, p,0.01), PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e41755