PDHK-2 and Fat Consumption in C. elegans Survival Figure 4. Expression pattern of lipases and their relative enzymatic activities in daf-2(e1370) controls and daf-2(e1370);pdhk-2 (tm3075) and daf-2(e1370);(tm3086) double mutants. (A) Lipase activity of daf-2(e1370);pdhk-2 double mutants at dauer day 1, normalized to daf- 2(e1370) dauer worms. (B) Lipase activity of daf-2(e1370);pdhk-2 double mutants at dauer day 10, normalized to daf-2(e1370) dauer worms. The values represent means [6SDs] from three independent experiments. (C) mRNA expression of lipases as determined by real-time qPCR in daf-2 (e1370) day 10 dauers. Data were normalized to daf-2(e1370) day 1 dauer worms. (D) Changes in lipase expression in daf-2(e1370);pdhk-2 double mutants compared to daf-2(e1370) in day 10 dauers. Data were normalized to day 1 dauer worms. Dauer worms were obtained from synchronized L1 stage worms after 60 hours at 25uC. The values represent means [6SDs] from two independent experiments. *p,0.05, **p,0.01 and ***p,0.001 compared with the control. doi:10.1371/journal.pone.0041755.g004 PDHK-2 activity under starved conditions. In contrast, PDHK-2 activity is unchanged in daf-16(mu86) or nhr-49(nr2041) mutants compared to daf-2(e1370) and N2 worms under well-fed condi- tions, consistent with the absence of a role for PDHK-2 in the context of an active glycolytic pathway. Although PDHK-2 has previously been shown to contribute to longevity in C. elegans [32,33], its role in MLS remains unresolved. For example, Kell et al. [32] reported that adult worms in which PDHK-2 was knocked down using RNA interference (RNAi) became sick or displayed vulval defects and exhibited shorter lifespans than control worms at 20uC. The authors concluded that the observed reduction in MLS caused by loss of PDHK-2 function might be the expected consequence, given that PDHK-2 plays an important role in energy consumption. However, Mouchiroud et al. [33] showed that inhibition or deficiency in pdhk-2 expression increased MLS by 20% compared to control worms at 20uC. Our results clearly favor the latter of these two contradictory findings, but only in the survival rate of long-term dauers (Figure 3F), in which a PDHK-2 deficiency appears to attenuate fat consumption through concomitant suppression of at least three major lipases (ATGL, HSL, and C07E3.9), leading to increased the survival rate of dauer larvae. As our results confirmed that PDHK-2 can be regulated by nutritional state, it may worth measuring the changes in lifespan of pdhk-2 mutants induced by dietary restriction or different food sources (e.g., HT115 or HB101 of E. Coli) in the hope that we can see better some effect of these two factors on the longevity of pdhk-2 mutant in the future. PDHK-2::GFP expression driven by the pdhk-2 promoter was mainly detected in head neurons (proximal rear side of IL-2 neurons) and the intestine (Figure 2, S3 and S4). The expression pattern of PDHK-2::GFP in head neurons was maintained in both the dauer stage and during starvation. It has been reported that PDHK-2 can control the nuclear localization of SKN-1, a representative regulator of dietary restriction in head neurons [32,34]. This observation suggests a potential link between the expression of PDHK-2 and SKN-1 in the head neuron. Although most lipases are expressed in the intestine [35], ATGL and HSL are known to be expressed in the nervous system, including in head neurons (Wormbase). Our data also show that PDHK-2 deficiency caused a decrease in ATGL, HSL and C07E3.9 (Figure 4D), consistent with this previous report. Thus, it is reasonable to speculate that the neuronal localization of PDHK-2 may be linked to metabolic control through the nervous system. In addition, PDHK-2 localization appears to be highly induced in muscle when worms are in starvation condition (Figure 2F). Muscle is one of the most energy consuming tissues which contain highest number of mitochondria. However, we could not find any clear evidence as to the correlation with PDHK-2 and mitochon- drial localization (data not shown). It is also possible that post- translational modifications of PDHK-2 (e.g., phosphorylation, acetylation or glycosylation) could change the localization of this enzyme according to the nutritional state. For example, the well- fed condition causes activation of IIS, which might consequently phosphorylate PDHK-2 or its binding proteins, leading to different localization of this protein. A characteristic morphological feature of dauer larvae is the presence of high-density fat granules in the intestine, which are very important for long-term survival during the dauer stage. Although studies have shown that L2 worms store fats before entering the dauer state in order to survive, how mobilization of these fats is regulated during this dormant period remains largely PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e41755