PDHK-2 and Fat Consumption in C. elegans Survival Figure 3. Functions of PDHK-2 in the dauer state. (A) Lifespan analysis of daf-2(e1370), daf-2(e1370);pdhk-2(tm3075), and daf-2(e1370);pdhk- 2(tm3086) mutant worms. (B) Oil Red O staining of daf-2(e1370), daf-2(e1370);pdhk-2(tm3075), and daf-2(e1370);pdhk-2(tm3086) worms at the day 1 adult stage. (C) Oil Red O staining of daf-2(e1370), daf-2(e1370);pdhk-2(tm3075), and daf-2(e1370);pdhk-2(tm3086) worms at the day 1 dauer stage. (D) Oil Red O staining of daf-2(e1370), daf-2(e1370);pdhk-2(tm3075), and daf-2(e1370);pdhk-2(tm3086) worms at the day 10 dauer stage. (E) TG content in daf-2(e1370), daf-2(e1370);pdhk-2(tm3075), and daf-2(e1370);pdhk-2(tm3086) worms at day 1 and day 10 of the dauer stage. The values represent means [6SDs] from three independent experiments. *p,0.05 and **p,0.001 compared with the control (F) Dauer survival assays performed on daf- 2(e1370), daf-2(e1370);pdhk-2(tm3075), daf-2(e1370);pdhk-2(tm3086), and daf-7(e1372) mutant worms. Dauer worms were obtained from synchronized L1 stage worms after 60 hours at 25uC. The values represent means [6SDs] from five independent experiments. Scale bar: 50 mm. doi:10.1371/journal.pone.0041755.g003 a 15% survival rate when worms were subjected to 1% SDS treatment. Thus, PDHK-2 overexpression caused both an increase in lipase expression and abnormal dauer formation, although the relationship between these two events is not clear at present. It is also not clear whether overexpression of PDHK-2 caused the activation of lipases that consequently led to this aberrant dauer formation and lifecycle. Discussion On the basis of the results presented here, we propose the following metabolic axis: PDHK-2 deficiencyRdecreased lipase- mediated fat consumptionRincreased survival rate under adverse nutritional conditions (i.e., dauer). This hypothesized relationship highlights an important regulatory role for PDHK-2 in starvation and the dauer state, where the glycolytic pathway has been shown to be less active [11,13], resulting in the greater availability of free fatty acids as fuel. Considered in this context, our results suggest that, as long as the glycolytic pathway is active, fatty acid mobilization does not take place; accordingly, PDHK-2 would not appear to play any role under these circumstances. Our results further suggest that PDHK-2 is highly regulated by both developmental stage (i.e., dauer) and nutritional conditions, which affect PDHK-2 localization and protein distribution, as well as transcriptional activation. This latter effect is mediated by DAF-16 and NHR-49; thus, DAF-16 or NHR-49 deficiencies affect PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e41755