PDHK-2 Deficiency Is Associated with Attenuation of Lipase-Mediated Fat Consumption for the Increased Survival of <em>Caenorhabditis elegans</em> Dauers

<div><p>In <em>Caenorhabditis elegans</em>, slow fat consumption has been suggested to contribute to the extension of the survival rate during nutritionally adverse conditions. Here, we investigated the potential role of pyruvate dehydrogenase kinase (PDHK)-2, the <em>C. elegans</em> homolog of mammalian PDK, effects on fat metabolism under nutritional conditions. PDHK-2 was expressed at low levels under well-fed conditions but was highly induced during long-term starvation and in the dauer state. This increase in <em>pdhk-2</em> expression was regulated by both DAF-16 and NHR-49. Dauer-specific induction of PDHK-2 was abolished upon entry into the post-dauer stage. Interestingly, in the long-term dauer state, stored fat levels were higher in <em>daf-2(e1370);pdhk-2</em> double mutants than in <em>daf-2(e1370)</em>, suggesting a positive relationship between PDHK-2 activity and fat consumption. PDHK-2 deficiency has been shown to lead to greater preservation of residual fats, which would be predicted to contribute to survival during the dauer state. A test of this prediction showed that the survival rates of <em>daf-2(e1370);pdhk-2(tm3075)</em> and <em>daf-2(e1370);pdhk-2(tm3086)</em> double mutants were higher than that of <em>daf-2(e1370)</em>, suggesting that loss of either the ATP-binding domain <em>(tm3075)</em> or branched chain keto-acid dehydrogenase kinase domain <em>(tm3086)</em> of PDHK-2 leads to reduced fat consumption and thus favors increased dauer survival. This attenuated fat consumption in the long-term dauer state of <em>C. elegans daf-2 (e1370);pdhk-2</em> mutants was associated with concomitant down-regulation of the lipases ATGL (adipose triglyceride lipase), HSL (hormone-sensitive lipase), and C07E3.9 (phospholipase). In contrast, PDHK-2 overexpression in wild-type starved worms induced lipase expression and promoted abnormal dauer formation. Thus, we propose that PDHK-2 serves as a molecular bridge, connecting fat metabolism and survival under nutritionally adverse conditions in <em>C. elegans</em>.</p> </div

Expression pattern of lipases and their relative enzymatic activities in <i>daf-2(e1370)</i> controls and <i>daf-2(e1370)</i>;<i>pdhk-2 (tm3075) and daf-2(e1370);(tm3086)</i> double mutants.

<p>(<b>A</b>) Lipase activity of <i>daf-2(e1370);pdhk-2</i> double mutants at dauer day 1, normalized to <i>daf-2(e1370)</i> dauer worms. (<b>B</b>) Lipase activity of <i>daf-2(e1370);pdhk-2</i> double mutants at dauer day 10, normalized to <i>daf-2(e1370)</i> dauer worms. The values represent means [±SDs] from three independent experiments. (<b>C</b>) mRNA expression of lipases as determined by real-time qPCR in <i>daf-2 (e1370)</i> day 10 dauers. Data were normalized to <i>daf-2(e1370)</i> day 1 dauer worms. (<b>D</b>) Changes in lipase expression in <i>daf-2(e1370);pdhk-2</i> double mutants compared to <i>daf-2(e1370)</i> 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 25°C. The values represent means [±SDs] from two independent experiments. *<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001 compared with the control.</p

Functions of PDHK-2 in the dauer state.

<p>(<b>A</b>) Lifespan analysis of <i>daf-2(e1370)</i>, <i>daf-2(e1370)</i>;<i>pdhk-2(tm3075)</i>, and <i>daf-2(e1370);pdhk-2(tm3086)</i> mutant worms. (<b>B</b>) Oil Red O staining of <i>daf-2(e1370)</i>, <i>daf-2(e1370)</i>;<i>pdhk-2(tm3075)</i>, and <i>daf-2(e1370);pdhk-2(tm3086)</i> worms at the day 1 adult stage. (<b>C</b>) Oil Red O staining of <i>daf-2(e1370)</i>, <i>daf-2(e1370)</i>;<i>pdhk-2(tm3075)</i>, and <i>daf-2(e1370);pdhk-2(tm3086)</i> worms at the day 1 dauer stage. (<b>D</b>) Oil Red O staining of <i>daf-2(e1370)</i>, <i>daf-2(e1370)</i>;<i>pdhk-2(tm3075)</i>, and <i>daf-2(e1370);pdhk-2(tm3086)</i> worms at the day 10 dauer stage. (<b>E</b>) TG content in <i>daf-2(e1370)</i>, <i>daf-2(e1370)</i>;<i>pdhk-2(tm3075)</i>, and <i>daf-2(e1370);pdhk-2(tm3086)</i> worms at day 1 and day 10 of the dauer stage. The values represent means [±SDs] from three independent experiments. *<i>p</i><0.05 and **<i>p</i><0.001 compared with the control (<b>F</b>) Dauer survival assays performed on <i>daf-2(e1370)</i>, <i>daf-2(e1370)</i>;<i>pdhk-2(tm3075)</i>, <i>daf-2(e1370);pdhk-2(tm3086)</i>, and <i>daf-7(e1372)</i> mutant worms. Dauer worms were obtained from synchronized L1 stage worms after 60 hours at 25°C. The values represent means [±SDs] from five independent experiments. Scale bar: 50 µm.</p

PDHK-2::GFP is highly expressed in starvation and dauer stages.

<p>(<b>A</b>) <i>pdhk-2</i>p::PDHK-2::GFP localization in the N2 adult stage. (<b>B</b>) <i>pdhk-2</i>p::PDHK-2::GFP localization in the L3 stage after good nutrition. (<b>C</b>) <i>pdhk-2</i>p::PDHK-2::GFP localization in the L3 stage after 12-hour of starvation. (<b>D</b>) <i>pdhk-2</i>p::PDHK-2::GFP localization in the dauer stage after 7 days of starvation. (<b>E</b>) Intestinal expression of <i>pdhk-2</i>p::PDHK-2::GFP under well-fed conditions at the L4 stage. (<b>F</b>) Muscle expression of <i>pdhk-2</i>p::PDHK-2::GFP in worms starved for 12-hour at the L4 stage. Scale bar: 50 µm.</p

<i>pdhk-2</i> mRNA levels under various conditions.

<p>Changes in <i>pdhk-2</i> gene expression were determined by real-time qPCR. (<b>A</b>) Expression levels of <i>pdhk-2</i> in <i>daf-2(e1370)</i>, <i>daf-7(e1372)</i>, <i>nhr-49(nr2041)</i>, and <i>daf-16(mu86)</i> mutants from well-fed L4 stage worms. The data were normalized to N2 well-fed L4-stage worms. (<b>B</b>) Expression levels of <i>pdhk-2</i> in N2 worms starved for 2, 6 and 12 hours from the L4 stage, and in dauer and post-dauer stage worms. Dauer worms were obtained after starving for 7 days at 20°C, whereas post-dauer worms were produced by feeding dauer worms for 12-hour at 20°C. The values were normalized to well-fed conditions at the L4 stage worms. Worms maintained under well-fed conditions at the L3 stage were used as controls for the dauer stage group. (<b>C</b>) Expression levels of <i>pdhk-2</i> in <i>daf-2(e1370) and daf-7(e1372)</i> mutants in the pre-dauer (L1+36 hours) and dauer (L1+60 hours) states at 25°C. The values were normalized to pre-dauer (L1+36 hours) worms. (<b>D</b>) Expression levels of <i>pdhk-2</i>, <i>nhr-49</i> and <i>daf-16</i> in N2, <i>daf-16(mu86)</i> and <i>nhr-49(nr2041)</i> worms under 12-hour starvation conditions. In each experiment, mutant worms maintained under well-fed conditions at the L4 stage were used as controls for the starvation group. The values represent means [±SDs] from three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001 compared with the control.</p