variety of organisms via AMP-activated protein kinase (AMPK) function [14,22]. In mammals, pyruvate dehydrogenase kinase-4 (PDK-4) is important in regulating pyruvate dehydrogenase during starvation [23–25]. In this study, we investigated how the pyruvate dehydrogenase kinase, PDHK-2, the C. elegans homolog of mammalian PDK-4 [23], contributes to the survival of C. elegans under the nutritionally adverse conditions. To this end, we used two different C. elegans pdhk-2 mutants—pdhk-2(tm3075), which lacks the C-terminal ATP- binding domain, and pdhk-2(tm3086), which lacks the N-terminal mitochondrial branched chain a-ketoacid dehydrogenase (BCDH) kinase domain (Figure S1)—to assess changes in gene expression and enzymatic activity under the nutritional extremes of well-fed and 12-hour starvation, as well as in the dauer state. We then correlated these changes with regulation of fatty acid metabolism and dauer survival rate. Here, we report that PDHK-2 deficiency appears to contribute to the preservation of energy through down- regulation of the activity of a subset of lipases, namely ATGL (adipose triglyceride lipase), HSL (hormone-sensitive lipase), and C07E3.9 (phospholipase), under nutritionally adverse conditions, and thereby promotes the long-term survival of C. elegans in the dauer stage. Results PDHK-2 expression is highly induced in starvation and in the dauer state To understand how PDHK-2 activity is related to longevity and fat metabolism, we first measured the MLS of two pdhk-2 deletion mutants (Figure S2). In well-fed conditions, mutant strains showed no significant changes in MLS (tm3075, 20.561.4 days; tm3086, 19.561.0 days) compared to the wild-type N2 strain (20.561.8 days) (Figure S2A). Second, we measured fat density and TG content using Oil Red O staining and TG assays, respectively, and found no significant differences between N2 worms and the pdhk-2 mutants, tm3075 and tm3086 (Figure S2B, C). Because DAF-2 and DAF-7 are known to play key roles in the IIS and tumor growth factor-b (TGF-b) signaling pathways, respectively, which are critical for the longevity and dauer formation, we also measured pdhk-2 mRNA levels in daf-2(e1370) and daf-7(e1372) mutant worms using total mRNA prepared from L4-stage worms grown under well-fed conditions. We found that the expression level of pdhk-2 did not significantly differ in daf-2(e1370) and daf-7(e1372) mutant worms compared to N2 worms (Figure 1A). Similarly, pdhk-2 expression levels were unchanged in nhr-49(nr2041) mutants, which lack a nuclear hormone receptor involved in fatty acid metabolism, and daf-16(mu86) mutants, which lack the IIS downstream transcriptional regulator, DAF-16 (Figure 1A). Because the expression of mammalian PDK-4 is known to increase during fasting [26], we examined changes in pdhk-2 expression in N2 worms grown under starvation conditions. We found a substantial increase (4.2-fold) in pdhk-2 gene expression in 12-hour starved groups compared to well-fed N2 worms (Figure 1B). Furthermore, pdhk-2 expression was increased by 8- fold in the dauer state (induced by 7 days starvation of N2 on NGM plates at 20uC) compared to controls (Figure 1B). This dauer-specific induction of pdhk-2 gene expression was abolished upon entry into the post-dauer stage (i.e., dauer recovery under well-fed conditions; Figure 1B). Similarly, as shown in Figure 1C, both daf-2(e1370) and daf-7(e1372) mutants also showed enhanced pdhk-2 expression (2.9-fold and 3.3-fold, respectively) upon entry into the complete dauer state (L1+60 hours at 25uC; see ‘Materials and Methods’). This induction of pdhk-2 expression in starvation PDHK-2 and Fat Consumption in C. elegans Survival and in the dauer state was not observed in either of the pdhk-2 mutants, tm3075 and tm3086 (data not shown). PDHK-2 expression is regulated by DAF-16 and NHR-49 in the starved state It has been previously reported that the mammalian F-box protein, FOXO1, the C. elegans homolog of DAF-16, induces PDK-4 expression in mammalian skeletal muscle during starvation [27]. Further, mammalian PDK-4 is known to be regulated by peroxisome proliferator-activated receptor (PPAR)-a, a functional homolog of C. elegans NHR-49 [28,29] that is essential for the fasting-dependent induction of genes involved in converting fat stores into energy [30]. To investigate whether PDHK-2 is regulated by DAF-16 and/or NHR-49 in C. elegans, we measured pdhk-2 mRNA levels in these mutants and N2 control worms that had been maintained under 12-hour starvation conditions. In contrast to the well-fed state, where mutant worms showed no significant changes in pdhk-2 expression compared to N2 controls (Figure 1A), with long-term starvation, the relative expression levels of pdhk-2 were more than 2.0-fold lower in daf-16(mu86) and nhr-49(nr2041) mutants than in N2 controls (1.7 and 2.1 for daf- 16(mu86) and nhr-49(nr2041), respectively, vs. 4.2 for N2; Figure 1D, left set of bars), indicating that both DAF-16 and NHR-49 act as activators of pdhk-2 expression under long-term starvation conditions. To examine the epistatic relationship between DAF-16 and NHR-49 with respect to PDHK-2 activation, we measured the expression levels of daf-16 and nhr- 49 mRNA in N2 controls and daf-16(mu86) and nhr-49(nr2041) mutant strains under 12-hour starvation conditions. Whereas the expression of nhr-49 in daf-16(mu86) mutant was reduced compared to N2 with 12-hour starvation (1.2 vs. 2.9; Figure 1D, middle bars), the levels of daf-16 expression in the nhr-49(nr2041) mutant were not significantly changed (Figure 1D, right bars, p = 0.1695). These results suggest that DAF-16 epistatically regulates NHR-49, while both DAF-16 and NHR-49 induce PDHK-2 under starvation conditions. Localization of PDHK-2 is changed during starvation To investigate if protein expression of PDHK-2 is changed by nutritional status, we first examined PDHK-2 localization in the well-fed state using the transgenic lines pdhk-2 promoter::GFP (pdhk-2p::GFP) and pdhk-2p::PDHK-2::GFP (PDHK-2::GFP). We found that pdhk-2p::GFP (Figure S3) and PDHK-2::GFP (Figure 2A and Figure S4), respectively, are expressed mainly in the head neurons, pharynx, intestinal nuclei and intestine. The expression levels of both of pdhk-2 mRNA and PDHK-2::GFP, which reflect PDHK-2 mRNA and protein expression, were increased under 12-hour starvation conditions (Figure 2; compare panel B with panel C) and in the dauer state (Figure 2; compare panel B with panel D). PDHK-2::GFP was ubiquitously expressed in the dauer state (Figure 2D). Interestingly, PDHK-2::GFP was dispersed around the nuclei of intestinal cells in L4 worms under well-fed conditions (Figure 2E). However PDHK-2::GFP exhibited a punctate fluorescent pattern in the muscle of worms that had been maintained under 12-hour starvation conditions (Figure 2F). These results suggest that localization of PDHK-2 is influenced by nutritional state (Figure 2E). PDHK-2 deficiency is associated with attenuated fat consumption and leads to increased survival rate of dauer larvae Animals store fats in adipose tissue (intestines in C. elegans) primarily in the form of TGs, which are usually hydrolyzed by TG PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e41755