Diacylglycerol lipase regulates lifespan and oxidative stress response by inversely modulating TOR signaling in Drosophila and C. elegans

Abstract

Target of rapamycin (TOR) signaling is a nutrient-sensing pathway controlling metabolism and lifespan. Although TOR signaling can be activated by a metabolite of diacylglycerol (DAG), phosphatidic acid (PA), the precise genetic mechanism through which DAG metabolism influences lifespan remains unknown. DAG is metabolized to either PA via the action of DAG kinase or 2-arachidonoyl-sn-glycerol by diacylglycerol lipase (DAGL). Here, we report that in Drosophila and Caenorhabditis elegans, overexpression of diacylglycerol lipase (DAGL/inaE/dagl-1) or knockdown of diacylglycerol kinase (DGK/rdgA/dgk-5) extends lifespan and enhances response to oxidative stress. Phosphorylated S6 kinase (p-S6K) levels are reduced following these manipulations, implying the involvement of TOR signaling. Conversely, DAGL/inaE/dagl-1 mutants exhibit shortened lifespan, reduced tolerance to oxidative stress, and elevated levels of p-S6K. Additional results from genetic interaction studies are consistent with the hypothesis that DAG metabolism interacts with TOR and S6K signaling to affect longevity and oxidative stress resistance. These findings highlight conserved metabolic and genetic pathways that regulate aging.

Keywords: S6 kinase; aging; diacylglycerol; diacylglycerol kinase; metabolism; phosphatidic acid.

Figure 1

DAGL/inaE expression regulates lifespan and oxidative stress response, and negatively correlates with phosphorylated S6 kinase (p-S6K) levels in Drosophila (A–D). The Drosophila DAGL/inaE^EP1101 mutant, which exhibits up-regulation of DAGL/inaE, shows extended lifespan, enhanced oxidative stress response and reduced levels of p-S6K. (A) Lifespan of DAGL/inaE^EP1101 (mean = 64 d, n = 306, red line) and w¹¹¹⁸ (mean = 37 d, n = 273, blue line). (B) Survival under 10 mm paraquat-induced oxidative stress of DAGL/inaE^EP1101 (mean = 74 h, n = 87, red line) and w¹¹¹⁸ (mean = 44 h, n = 69, blue line). (C) RT-PCR analysis shows a nearly 200% increase in DAGL/inaE levels in DAGL/inaE^EP1101 compared to that of w¹¹¹⁸. (D) Levels of p-S6K are decreased in DAGL/inaE^EP1101 compared to that of w¹¹¹⁸ in 3-d and 20-d old flies. (E–H) The DAGL/inaE^KG08585 mutant, which exhibits down-regulation of DAGL/inaE, shows shortened lifespan, reduced oxidative stress resistance and increased levels of p-S6K. (E) Lifespan of DAGL/inaE^KG08585 (mean = 21 d, n = 254, red line) and w¹¹¹⁸ (mean = 41 d, n = 300, blue line). (F) Survival under 10 mm paraquat-induced oxidative stress of DAGL/inaE^KG08585 (mean = 37 h, n = 100, red line) and w¹¹¹⁸ (mean = 55 h, n = 100, blue line). (G) RT-PCR analysis shows a 50% decrease in DAGL/inaE levels in DAGL/inaE^KG08585 compared to that of w¹¹¹⁸. RT-PCR results are normalized to rp49 as an internal control (C, G). (H) Levels of p-S6K are increased in DAGL/inaE^KG08585 compared to that of w¹¹¹⁸ in 3-d and 20-d old flies. α-actin was used as an internal control (D, H).

Figure 2

Genetic interaction between DAGL/inaE, rdgA, and S6K on lifespan. (A) Overexpression of DAGL/inaE driven by GMR-Gal4 (GMR-Gal4 > UAS-DAGL/inaE, mean = 49 d, n = 206) extends lifespan compared to controls harboring either genetic element alone (GMR-Gal4/+, mean = 34 d, n = 258; UAS-DAGL/inaE/+, mean = 42 d, n = 292). (B) Knockdown of rdgA by RNAi (GMR-Gal4>UAS-rdgA^RNAi, mean = 50 d, n = 62) also extends lifespan compared to controls (GMR-Gal4/+, mean = 34 d, n = 258; UAS-rdgA^RNAi/+, mean = 44, n = 173). (C) Lifespan extension by simultaneous overexpression of DAGL/inaE and knockdown of rdgA (GMR-Gal4>UAS-DAGL/inaE /UAS-rdgA^RNAi, mean = 48 d, n = 203) is similar to either manipulation alone (GMR-Gal4>UAS-DAGL/inaE, mean = 49 d, n = 206; GMR-Gal4>UAS-rdgA^RNAi, mean = 50 d, n = 62). (D) Overexpression of DAGL/inaE driven by da-Gal4 (da-Gal4 > UAS-DAGL/inaE, mean = 36 d, n = 210) extends lifespan compared to controls (da-Gal4/+, mean = 29 d, n = 209; UAS-DAGL/inaE/+, mean = 30 d, n = 232) at 29 °C. (E) Overexpression of S6K^KQ, a dominant-negative form of S6K, (da-Gal4 > UAS-S6K^KQ, mean = 35 d, n = 50) also extends lifespan compared to controls (da-Gal4/+, mean = 29 d, n = 209; UAS-S6K^KQ/+, mean = 26 d, n = 129) at 29 °C. (F) Lifespan extension by simultaneous overexpression of both DAGL/inaE and S6K^KQ (da-Gal4 > UAS-DAGL/inaE/UAS-S6K^KQ, mean = 33 d, n = 76) does not further extend lifespan compared to either manipulation alone (da-Gal4 > UAS-DAGL/inaE, mean = 36 d, n = 210) or S6K^KQ (da-Gal4 > UAS-S6K^KQ, mean = 35 d, n = 50) at 29 °C.

Figure 3

dagl-1 expression regulates lifespan and negatively correlates with levels of p-S6K in C. elegans. (A) Two independent transgenic lines that overexpress dagl-1 (N2; Ex[Pdpy::dagl-1::GFP](3), red line, and N2; Ex[Pdpy::dagl-1::GFP](4), green line) show extended lifespan compared to the control (N2; Ex[Pdpy::GFP], blue line). (B) N2 worms treated with RNAi against either 5′ (dagl-1(RNAi-1)) or 3′ (dagl-1(RNAi-2)) coding sequence of dagl-1 display shortened lifespan compared to the empty vector (EV) control. (C, D) The two dagl-1 deletion mutants, dagl-1(tm2908) and dagl-1(tm3026), exhibit reduced lifespan compared to the control N2. (E, F) Shortened lifespan of dagl-1(tm2908) and dagl-1(tm3026) can be rescued by transgenic overexpression of dagl-1. See also Table S3. (G) Western blot shows elevated levels of p-S6K in dagl-1(tm2908) and dagl-1(tm3026). β-actin was used as an internal control. (H) Elevated levels of p-S6K in dagl-1(tm2908) and dagl-1(tm3026) are reduced by transgenic overexpression of dagl-1. Western blots are shown in Fig. S8A.

Figure 4

Knockdown of dgk-5, daf-15, or let-363 rescues the shortened lifespan and elevated p-S6K levels in dagl-1 mutants. (A) Elevated levels of p-S6K in dagl-1(tm2908) and dagl-1(tm3026) are reduced by RNAi knockdown of dgk-5. (B) Shortened lifespan of dagl-1(tm2908) and dagl-1(tm3026) is rescued by RNAi knockdown of dgk-5. (C) RNAi knockdown of daf-15 or let-363 also reverts the elevated levels of p-S6K observed in dagl-1(tm2908) and dagl-1(tm3026). (D–G) Shortened lifespan of dagl-1 mutants is also rescued by RNAi knockdown of daf-15 or let-363. See also Table S5. All western blots are shown in Fig. S8B and C. (H) Model for DAGL/inaE/dagl-1 in regulation of lifespan in Drosophila and C. elegans. DAGL/inaE/dagl-1 overexpression reduces TOR signaling and p-S6K levels to slow aging (red arrows). Hypomorphs of DAGL/inaE/dagl-1 increase TOR signaling and p-S6K levels to accelerate aging (green arrows).