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

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

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

Quantitative Profiling Identifies Potential Regulatory Proteins Involved in Development from Dauer Stage to L4 Stage in <i>Caenorhabditis elegans</i>

When <i>Caenorhabditis elegans</i> encounters unfavorable growth conditions, it enters the dauer stage, an alternative L3 developmental period. A dauer larva resumes larval development to the normal L4 stage by uncharacterized postdauer reprogramming (PDR) when growth conditions become more favorable. During this transition period, certain heterochronic genes involved in controlling the proper sequence of developmental events are known to act, with their mutations suppressing the Muv (multivulva) phenotype in <i>C. elegans.</i> To identify the specific proteins in which the Muv phenotype is highly suppressed, quantitative proteomic analysis with iTRAQ labeling of samples obtained from worms at L1 + 30 h (for continuous development [CD]) and dauer recovery +3 h (for postdauer development [PD]) was carried out to detect changes in protein abundance in the CD and PD states of both N2 and <i>lin-28­(n719)</i>. Of the 1661 unique proteins identified with <i>a</i> < 1% false discovery rate at the peptide level, we selected 58 proteins exhibiting ≥2-fold up-regulation or ≥2-fold down-regulation in the PD state and analyzed the Gene Ontology terms. RNAi assays against 15 selected up-regulated genes showed that seven genes were predicted to be involved in higher Muv phenotype (<i>p</i> < 0.05) in <i>lin-28­(n791)</i>, which is not seen in N2. Specifically, two genes, K08H10.1 and W05H9.1, displayed not only the highest rate (%) of Muv phenotype in the RNAi assay but also the dauer-specific mRNA expression, indicating that these genes may be required for PDR, leading to the very early onset of dauer recovery. Thus, our proteomic approach identifies and quantitates the regulatory proteins potentially involved in PDR in <i>C. elegans</i>, which safeguards the overall lifecycle in response to environmental changes

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

Quantitative Profiling Identifies Potential Regulatory Proteins Involved in Development from Dauer Stage to L4 Stage in <i>Caenorhabditis elegans</i>

When <i>Caenorhabditis elegans</i> encounters unfavorable growth conditions, it enters the dauer stage, an alternative L3 developmental period. A dauer larva resumes larval development to the normal L4 stage by uncharacterized postdauer reprogramming (PDR) when growth conditions become more favorable. During this transition period, certain heterochronic genes involved in controlling the proper sequence of developmental events are known to act, with their mutations suppressing the Muv (multivulva) phenotype in <i>C. elegans.</i> To identify the specific proteins in which the Muv phenotype is highly suppressed, quantitative proteomic analysis with iTRAQ labeling of samples obtained from worms at L1 + 30 h (for continuous development [CD]) and dauer recovery +3 h (for postdauer development [PD]) was carried out to detect changes in protein abundance in the CD and PD states of both N2 and <i>lin-28­(n719)</i>. Of the 1661 unique proteins identified with <i>a</i> < 1% false discovery rate at the peptide level, we selected 58 proteins exhibiting ≥2-fold up-regulation or ≥2-fold down-regulation in the PD state and analyzed the Gene Ontology terms. RNAi assays against 15 selected up-regulated genes showed that seven genes were predicted to be involved in higher Muv phenotype (<i>p</i> < 0.05) in <i>lin-28­(n791)</i>, which is not seen in N2. Specifically, two genes, K08H10.1 and W05H9.1, displayed not only the highest rate (%) of Muv phenotype in the RNAi assay but also the dauer-specific mRNA expression, indicating that these genes may be required for PDR, leading to the very early onset of dauer recovery. Thus, our proteomic approach identifies and quantitates the regulatory proteins potentially involved in PDR in <i>C. elegans</i>, which safeguards the overall lifecycle in response to environmental changes

<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