PDHK-2 and Fat Consumption in C. elegans Survival Figure 2. PDHK-2::GFP is highly expressed in starvation and dauer stages. (A) pdhk-2p::PDHK-2::GFP localization in the N2 adult stage. (B) pdhk-2p::PDHK-2::GFP localization in the L3 stage after good nutrition. (C) pdhk-2p::PDHK-2::GFP localization in the L3 stage after 12-hour of starvation. (D) pdhk-2p::PDHK-2::GFP localization in the dauer stage after 7 days of starvation. (E) Intestinal expression of pdhk-2p::PDHK-2::GFP under well-fed conditions at the L4 stage. (F) Muscle expression of pdhk-2p::PDHK-2::GFP in worms starved for 12-hour at the L4 stage. Scale bar: 50 mm. doi:10.1371/journal.pone.0041755.g002 daf-2(e1370);pdhk-2(tm3086) (16.560.97 days, p,0.001) double mutants and daf-7(e1372) (18.860.89 days p,0.0001) compared to daf-2(e1370) controls (11.461.76 days; Figure 3F). This increased survival rate, which ranged from 35% to 45%, appeared to be attributable to the higher levels of residual fat in the bodies of these double mutants, suggesting that both ATP-binding and BCDH domains may be associated with fat consumption. Thus, disruption of either leads to the reduced fat consumption that is necessary for survival during the long-term dauer state. PDHK-2 deficiency is associated with attenuated lipase activity Our results seem to suggest that PDHK-2 deficiency causes suppression of lipolysis, which is usually mediated by lipases. To test how lipase activity is influenced by PDHK-2 deficiency, we measured the enzymatic activity of total cellular lipases in the daf- 2(e1370);pdhk-2(tm3075) and daf-2(e1370);pdhk-2(tm3086) double mutants at different dauer-state durations. In both short-term and long-term dauer groups, total lipase activity was substantially reduced in the double mutants (28–43%) compared to daf-2(e1370) controls (Figure 4A, B), confirming a strong positive correlation between lipase and PDHK-2 activity. There is very minor difference in lipase activity between the short-term dauer and long-term dauer of daf-2(e1370) (data not shown). Loss of either the ATP-binding (tm3075) or BCDH (tm3086) domain of PDHK-2 in daf-2;pdhk-2 double mutants appeared to similarly contribute to this concomitant down-regulation of PDHK-2 and lipase activity (Figure 4A, B). To identify which lipase(s) might be involved in mediating fat catabolism in response to dauer state duration (short- vs. long-term), we examined the expression of eight currently identified and predicted lipases. We found highly induced expression of ATGL (1.6-fold), HSL (2.5-fold), and C07E3.9 (2.6-fold) in daf-2(e1370) long-term (day 10) dauers (Figure 4C). Notably, the increased expression of these three lipases was abolished in daf-2(e1370);pdhk-2(tm3075) and daf-2(e1370);pdhk- 2(tm3086) double mutants during the long-term dauer state, indicating that these lipases may respond specifically to the suppression of PDHK-2 activity (Figure 4D). Overexpression of PDHK-2 induces abnormal dauer formation To investigate the effect of PDHK-2 overexpression (PDHK-2/ OE) on the lifecycle of C. elegans, we microinjected pdhk- 2p::PDHK-2::GFP into N2 worms and the pdhk-2 mutants, tm3075 and tm3086; N2 injected with pdhk-2p::GFP was used as a control. After growing under well-fed or starved conditions, we examined MLS and dauer formation rate. Well-fed worms showed no significant difference in MLS following PDHK-2 overexpres- sion: N2 control (pdhk-2p::GFP), 16.462.1 days; N2;PDHK-2/ OE, 17.762.0 days; tm3075;PDHK-2/OE, 18.561.5 days; and tm3086;PDHK-2/OE, 18.462.0 days (Figure 5A). In contrast, PDHK-2 overexpression in N2 caused an increase in the expression of most of lipases examined compared to control worms maintained under 12-hour starvation conditions (Figure 5B). Furthermore, when maintained under starvation conditions (.15 days at 25uC) or in daumone plate assays (data not shown) [31], daf-2(e1370) worms overexpressing PDHK-2 (daf- 2(e1370;PDHK-2/OE) exhibited a reduction in the rate of dauer formation compared to daf-2(e1370) control worms (daf- 2(e1370);pdhk-2p::GFP). That is, most daf-2(e1370) worms at the L1 stage typically entered dauer formation within 3 days (.60 hours) at 25uC, whereas transgenic daf-2(e1370);PDHK-2/ OE worms displayed slow growth, abnormal dauer formation, and even bypassed the dauer stage (Figure 5C). We also observed only PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e41755