PDHK-2 and Fat Consumption in C. elegans Survival Figure 5. Effect of PDHK-2 overexpression. (A) Lifespan analysis of PDHK-2 overexpression in N2 wild-type. pdhk-2p::PDHK-2::GFP was microinjected into N2 worms and the pdhk-2 mutants, tm3075 and tm3086; N2 injected with pdhk-2p::GFP was used as a control. (B) Changes in lipase expression in PDHK-2-overexpressing N2 worms after 12-hour under starvation conditions. All data were normalized to control worms under 12-hour starvation conditions. *p,0.05, **p,0.01 and ***p,0.001 compared with the control. The values represent means [6SDs] from two independent experiments. (C) Effect of PDHK-2 overexpression in daf-2(e1370) dauer stage worms. In the lower panel, normal dauer worms (on the left) do not express GFP, whereas worms exhibiting abnormal dauer formation do express GFP (on the right). Scale bar: 50 mm. (D) Role of PDHK-2 under nutritionally adverse conditions in C. elegans. Shown is the proposed role of PDHK-2 in the maintenance of fat reservoirs in starved or dauer larvae, which depend on their fat supply for long-term survival. Nutrient signals act through DAF-16 and/or NHR-49 (or through a separate, currently unidentified mechanism) to regulate PDHK-2, which in turn appears to control the expression of lipases. doi:10.1371/journal.pone.0041755.g005 unknown. Since fat utilization is critical for long-term survival during the dauer state or starvation, our data suggest that PDHK- 2 may be involved in the fine-tuning of fat consumption by modulating lipase expression specifically in response to the adverse nutritional conditions, in which IIS and TGF-b signaling are inactive (Figure 1C) but DAF-16 is active. That is, under well-fed conditions, PDHK-2 activity does not seem to influence lifespan and fat content, regardless of the activity of major signaling pathways or effecter molecules (NHR-49, DAF-16). However, with long-term starvation or in the dauer state, DAF-16 and NHR-49 apparently work to up-regulate PDHK-2, which consequently stimulates major lipases (e.g., ATGL, HSL and C07E3.9), leading to a supply of fat-driven energy for survival (Figure 5D). Conversely, if PDHK-2 is defective, such as in pdhk- 2(tm3075) (this report), lipase expression is reduced (through an unknown mechanism), thereby inhibiting fat lipolysis and leading to sustained maintenance of a fat energy source necessary for survival. If PDHK-2 is also defective in dauer stage, lipase activity would also be reduced. In case of daf-2, there was essentially no change in lipase activity between dauer day 1 and day 10 (data not shown), indicating that different types of lipases might become activated during the dauer maintenance period (Figure 4C). In particular, PDHK-2 appears to control the expression of ATGL, HSL and C07E3.9, which may cause lipase activity lower than control (Figure 4D). One probable clue to the relationship between PDHK-2 deficiency and decreased lipase activity may be found in the intersection of AMPK signaling and lipases. Accordingly, as previously reported [14], the extended dauer state of C. elegans dauer larvae is maintained through the control of the LKB1/ AMPK pathway, which suppresses signals that promote lipase activity, resulting in the slow release of hypodermal fat. This observation is similar to our demonstration that mutation of pdhk-2 leads to reduced lipase activity and greater accumulation of fats in the body, which may contribute to the increased the survival rate of dauer larvae (Figure 3C–F). Whereas LKB1/AMPK signaling directly controls a specific target lipase (i.e., ATGL) [14], PDHK-2 appears to regulate a broad range of candidate lipases in the dauer state (Figure 4D) and exerts differential regulation of lipases depending on the nutritional condition (data not shown). This led us to speculate that there might be nutrition state-specific lipase regulation, as is the case for LIPL-4, which is closely associated with the autophagy process through which longevity can be controlled in germline-less animals [36]. This observation suggests the possible existence of additional systems for regulation of lipases in response to nutrient signals. For example, under conditions of PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e41755