dietary restriction, PDHK-2 appears to be associated with AMPK and other factors [33]. PDHK-2 deficiency, which causes the increased survival rate through suppression of lipase activity in dauer larvae, appears to have the opposite effect in non-dauer animals. Loss of germline and reduced daf-2 signaling were shown to synergistically induce the lipase K04A8.5 (LIPL-4) and decrease fat storage [35]. RNAi- mediated K04A8.5 knockdown partially suppressed the longevity of daf-2(e1370). In mammals, PDK-4 appears to be important in controlling diabetes and obesity [37], and its expression at the mRNA and protein levels is regulated by seasonal changes in hibernating animals, which require tight control of fuel selection. In C. elegans dauer larvae, fine-tuning of energy consumption is critical to the success of long-term survival through diapause. Similarly, the dauer state of C. elegans is regarded as a hibernating period in which accumulated fats are consumed to support survival throughout this non-aging state. PDHK-2 levels were dramatically increased in this dauer state, consistent with an essential role for PDHK-2 in dauer fat metabolism and survival. Lipases were highly expressed in PDHK-2-overexpressing N2 worms and daf-2(e1370) mutants leading to abnormal dauer formation through interruption of fat regulation, which is critical for dauer entry (Figure 5B, C). Although PDHK-2 overexpression showed no evident effect in the well-fed state, it negatively influenced dauer formation rate, perhaps due to over-consumption of lipids by highly activated lipase activity, leading to abnormal arrest (i.e., aberrant dauer formation). Thus, we propose that PDHK-2 serves as a molecular bridge, connecting fat metabolism and survival in coordination with DAF-16 and NHR-49 under nutritionally adverse conditions in C. elegans (Figure 5D). Materials and Methods Worm strains The C. elegans strains used in this study, N2 Bristol (wild type), daf-2(e1370), daf-7(e1372) and daf-16(mu86), were obtained from the Caenorhabditis Genetics Center. nhr-49(nr2041) was kindly provided by Carl Johnson and Nemapharm Pharmaceuticals. pdhk-2(tm3075) and pdhk-2(tm3086) were provided by the Japanese National Bioresource Project and were outcrossed six times to a wild-type background. Worms were cultured on nematode growth media (NGM) agar plates seeded with the Escherichia coli strain OP50, as previously described [38]. daf-2(e1370);pdhk-2(tm3075) and daf-2(e1370);pdhk-2(tm3086) double mutants were obtained through genetic crosses. Transgenic lines and plasmid construction The pdhk-2 promoter::green fluorescent protein (GFP) construct (pdhk-2p::GFP) was produced by cloning a 2.5-kb sequence upstream of the pdhk-2 start codon into the pPD95.75 vector using standard molecular biology protocols. The pdhk-2p::PDHK- 2::GFP construct contained a 1.2-kb pdhk-2 cDNA sequence (immediately prior to the stop codon) between the promoter sequence and GFP in the pPD95.75 vector. Constructs were microinjected at 50–100 ng/ml into N2, daf-2(e1370), and pdhk-2 mutant worms. GFP-expressing transgenic worms were selected from the progeny and analyzed. Starvation assay Starvation assays were performed as described previously [30]. For starved worms, embryos from bleached adults were allowed to hatch in S-basal medium. L1 larvae were added to NGM plates containing OP50 (well-fed condition). Worms were washed at the PDHK-2 and Fat Consumption in C. elegans Survival L4 stage using distilled water and placed onto assay plates (NGM without peptone) containing no bacteria for 2, 6 or 12 hours. Quantitative RT-PCR Total RNA was isolated from worms at various stages using the illustra RNAspin Mini RNA Isolation Kit (GE Healthcare). Total RNA was reverse transcribed with the Transcriptor First Strand cDNA Synthesis Kit (Roche) using oligo (dT) primers. Quantiti- tative polymerase chain reaction (qPCR) was performed using an MJ Research Chromo4 Detector with the QuantiTect SYBR Green PCR kit, as described by the manufacturer (Qiagen). b- actin was used as an internal control. The data are presented as means (6 standard deviations) of triplicate reactions from two or three independent experiments. Lifespan assay Lifespan assays were performed at 20uC under well-fed conditions. Embryos from bleached adults were allowed to hatch in S-basal medium to obtain highly synchronous L1 stage animals. L1 worms were then transferred to NGM plates and allowed to mature to the adult stage. Adult worms were transferred to fresh NGM plates each day during the period of reproduction. Worms that no longer responded to gentle prodding with a platinum wire were considered non-viable. Oil Red O staining Worms were stained with Oil Red O as described previously [39]. Worms were grown on NGM plates until the day 1 adult stage and then were washed three times with 16 phosphate- buffered saline (PBS). Worms were resuspended in 16 PBS and 26 MRWB (Modified Ruvkuns Witches Brew) buffer with paraformaldehyde for 1 hour at room temperature. In the case of dauer worms, additional freeze/thaw steps were performed using liquid nitrogen. Worms were washed with 16 PBS and then resuspended in 60% isopropanol and incubated for 15 minutes at room temperature. Isopropanol was removed, 60% Oil Red O stain was added, and worms were incubated overnight with rocking. Worms were washed with 16 PBS containing 0.01% Triton X-100 and visualization by microscopy. TG determination and lipase assay Proteins from well-fed and dauer worms were harvested as described previously [40]. Day 1 adult or dauer worms were washed three times with ice-cold M9 buffer. Worms were then flash frozen in liquid nitrogen without buffer. Approximately 100– 200 ml of worms were ground in a nitrogen-chilled mortar and pestle, then gathered in a reaction tube and kept on ice. PBS (16) was added to the powdered worms, and the extracts were sonicated and then centrifuged for 7 minutes at 12,0006g and 4uC to remove any insoluble matter. Protein concentration was measured by the Bradford assay. TG content was determined using a commercially available TG determination kit (Sigma- Aldrich). Lipase activity was determined using a QuantiChrom Kit (BioAssay Systems). Dauer survival assay Dauer worms were obtained by growing synchronized L1 stage worms (daf-2(e1370), daf-7(e1372) or daf-2;pdhk-2 double mutants) on assay plates (seeded with 2 ml OP50 culture) for 60 hours at 25uC. Dauer larvae thus obtained were washed five times with distilled water and diluted to 500 dauers/10 ml. A 500 ml suspension of dauer larvae was mixed in a 360u rotator (30 rpm) at 20uC so that dauer may continuously consume energy for PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e41755