ω-6 Polyunsaturated fatty acids extend life span through the activation of autophagy

Abstract

Adaptation to nutrient scarcity depends on the activation of metabolic programs to efficiently use internal reserves of energy. Activation of these programs in abundant food regimens can extend life span. However, the common molecular and metabolic changes that promote adaptation to nutritional stress and extend life span are mostly unknown. Here we present a response to fasting, enrichment of ω-6 polyunsaturated fatty acids (PUFAs), which promotes starvation resistance and extends Caenorhabditis elegans life span. Upon fasting, C. elegans induces the expression of a lipase, which in turn leads to an enrichment of ω-6 PUFAs. Supplementing C. elegans culture media with these ω-6 PUFAs increases their resistance to starvation and extends their life span in conditions of food abundance. Supplementation of C. elegans or human epithelial cells with these ω-6 PUFAs activates autophagy, a cell recycling mechanism that promotes starvation survival and slows aging. Inactivation of C. elegans autophagy components reverses the increase in life span conferred by supplementing the C. elegans diet with these fasting-enriched ω-6 PUFAs. We propose that the salubrious effects of dietary supplementation with ω-3/6 PUFAs (fish oils) that have emerged from epidemiological studies in humans may be due to a similar activation of autophagic programs.

Figure 1.

LIPL-4 overexpression mimics a physiological response that increases C. elegans resistance to starvation. (A) lipl-4 is transcriptionally up-regulated in fasting animals. lipl-4 mRNA abundance in fasting wild-type young adults relative to ad libitum feeding animals of the same age at different times from food withdrawal is depicted as mean ± SEM of two independent experiments. Statistical differences are depicted (*). (B) LIPL-4Ox animals express lipl-4 at twice the levels observed in fasted wild-type worms. lipl-4 mRNA levels in well-fed ges-1P∷LIPL-4∷SL2∷GFP young adults compared with control (nontransgenic siblings) is presented from three independent experiments, and all values are corrected for the efficiency of the primer pairs and normalized to ama-1 as internal control as well as to transcript levels in same age nontransgenic sibling animals. All quantitative RT–PCR (qRT–PCR) reactions were performed at least in triplicate. Median ± SEM of ΔΔCt is reported (Pfaffl 2001). (C) LIPL-4 overexpression promotes resistance to starvation. LIPL-4Ox and control (nontransgenic sibling [NTS]) hatchlings were sorted (COPAS Biosort) and independently grown on OP50 bacteria, then harvested as young L4 larvae and rotated at 20°C in 1 μM Sytox orange in S-basal. Every other day, aliquots were sorted to calculate the percentage of red (dead) worms. The average percentage of LIPL-4Ox animals alive on the same day that ∼50% of control worms were alive from three independent experiments ± SEM is depicted. See also representative L4 survival curve in Supplemental Figure S2. (D) Fasting and LIPL-4Ox lead to an enrichment of ω-3/6 PUFAs. Levels of AA and EPA relative to total FAMEs (Watts and Browse 2002; Van Gilst et al. 2005b) are depicted as the mean percentage ± SEM for wild-type well-fed and 8-h fasted and well-fed young adults (similar results were obtained when compared with nontransgenic siblings) (data not shown). (E) AA and DGLA, but not EPA, promote resistance to starvation. Ten micromolar AA, 20 μM EPA, 10 μM DGLA (concentrations of supplemented fatty acids that lead to levels of incorporation comparable with those observed in LIPL-4Ox animals) (see Supplemental Fig. S3), or an equal volume of 50% ethanol was added, and the percentage of animals alive was determined and presented as in C but using Sytox green to stain dead worms.

Figure 2.

ω-6 PUFAs activate autophagy in C. elegans. (A) LIPL-4Ox activates autophagy. LGG-1∷GFP signal in L3 animals overexpressing LIPL-4 from a constitutively highly expressed intestinal promoter versus control animals is shown (control corresponds to siblings not carrying LIPL-4∷TagRFP but carrying LGG-1∷GFP); exposure time = 100 msec. Quantification of LGG-1∷GFP punctae per anterior intestinal cell from at least three independent experiments is depicted as mean ± SEM using the same exposure time for LIPL-4∷TagRFP and control worms. At longer exposure times (500 msec), more LGG-1∷GFP punctae than at 100 msec can be observed in control animals; for comparison at 500 msec, see Supplemental Figure S5. For bleed-through and autofluorescence controls, see Supplemental Figure S4. (B) AA and DGLA activate autophagy in C. elegans. LGG-1∷GFP punctae per anterior intestinal cell in supplemented (50 μM) or mock-treated (50% ethanol) L3 animals are shown; data from three independent experiments are depicted as mean ± SEM. (C) fat-6, fat-7, or fat-1 inactivation is insufficient to activate autophagy in C. elegans. LGG-1∷RFP worms were egg-laid in fat-1, fat-6, fat-7, or vector control RNAi plates (qPCR analysis showed that the levels of expression of fat-1, fat-6, and fat-7 after RNAi treatment were 0.2 ± 0.1, 0.3 ± 0.05, 0.3 ± 0.1 of vector control treated animals, respectively). The pattern and intensity of the LGG-1∷RFP signal was scored in young adult worms. Representative control and fat RNAi-treated animals are shown. No difference was observed in the intensity or pattern of the expression of LGG-1∷RFP in treated animals when compared with empty vector-treated worms.

Figure 3.

ω-6 PUFAs activate autophagy in mammalian cells. (A) AA and DGLA supplementation leads to an increase in LC3II in HeLa cells. Fifty percent confluent cells were treated with 100 μM AA, DGLA, or EPA or mock-treated with an equal volume of 50% ethanol for 24 h. Representative Western blot analysis of endogenous LC3 in supplemented or mock-treated cells is shown, and quantification of LC3II (autophagy-specific form) signal intensity relative to actin from three independent experiments is depicted as mean ± SEM. (B) AA and DGLA activate autophagy in HeLa cells. The top row depicts HeLa cells stably expressing GFP-hLC3B grown in rich culture medium supplemented with 100 μM AA or DGLA or equal volume of ethanol and imaged after 30 h of total treatment. Quantification of GFP-hLC3B punctae is presented in the table below. The second row shows the increase in GFP-hLC3B punctae observed when the lysosomal inhibitors E64d (5 μg/mL) and pepstatin (10 μg/mL) were added to cells pretreated for 24 h with carrier or fatty acids and incubated for another 6 h with the inhibitors. Quantification of GFP-hLC3B punctae is presented in the table below. The third row shows lysosomal breakdown capacity in the presence of fatty acids as revealed by the cleavage and consequent dequenching of 10 μg/mL red DQ-BSA added after a 24-h treatment with fatty acids and incorporated and cleaved for 6 h. The fourth row shows that the GFP-hLC3B and red DQ-peptide signals overlap in the AA- and DGLA-treated cells (merges between the top and third row images are shown). For all experiments, images were taken after 30 h of total treatment. Images are representative from at least three independent experiments. Bars, 20 μm. The table shows GFP-hLC3B punctae quantification and statistical analyses for three independent experiments. Proportion of cells showing more than five GFP-hLC3B punctae ≥2 μm in the presence or absence of the lysosomal inhibitors is shown as percentages of the total number of cells (n) analyzed per treatment. The number of dots >2 μm per cell was quantified from at least 10 different fields using SPOT software. P-values relative to mock-treated cells are presented at the bottom of the columns. Protein aggregation control is shown in Supplemental Figure S7A, and p62/SQSTM1 degradation control is shown in Supplemental Figure S7B. (C) AA- and DGLA-triggered activation of autophagy in mouse embryonic fibroblasts (MEFs) is ATG16L1-dependent. The top row shows wild-type MEFs stably expressing GFP-hLC3B grown in rich culture medium supplemented with 100 μM AA or DGLA or equal volume of ethanol and imaged after 24 h of total treatment. The bottom row shows ATG16L1^−/− MEFs stably expressing GFP-hLC3B grown in rich culture medium supplemented with 100 μM AA or DGLA or equal volume of ethanol and imaged after 24 h of total treatment.

Figure 4.

ω-6 PUFAs extend life span through the activation of autophagy. (A) Autophagy contributes to LIPL-4Ox longevity. Representative survival curve of LIPL-4Ox or control (nontransgenic siblings) incubated at 20°C in OP50 plates and transferred as L4 larvae to vector control or RNAi against core autophagy genes is presented (mean life spans: control = 16.1, LIPL-4Ox = 19.0; P = 3.21 × 10⁻⁵). Mean life span and statistical significance of the differences compared with control of an independent experiment are presented in Supplemental Table S1A. (B) The autophagy transcriptional regulator PHA-4 (FoxA), but not the starvation regulator DAF-16 (FoxO), contributes to the LIPL-4Ox-extended life span phenotype. Representative survival curve of LIPL-4Ox (ges-1 promoter) or nontransgenic siblings (NTS) grown at 20°C in OP50 plates and transferred as L4 larvae to vector control, pha-4, or daf-16 RNAi is presented. Mean life span and statistical significance of the differences compared with control are presented in Supplemental Table S1B. (C) AA and DGLA extend C. elegans life span. Survival curves of worms grown in OP50 plates and transferred as L4 larvae to plates supplemented with ω-3/6 PUFAs or carrier are presented. Worms were incubated at 20°C and transferred every other day to fresh plates containing 10 μM AA, 10 μM DGLA, 20 μM EPA, or an equal volume of carrier until cessation of reproduction. Worms were then transferred every 7 d to maintain the strength of the treatments. Statistical analysis of an independent experiment is presented in Supplemental Table S2A. (D) AA and DGLA extend C. elegans life span through activation of autophagy. Survival curves of worms grown in OP50 plates, transferred as L4 larvae to RNAi plates supplemented with ω-3/6 PUFAs or carrier, and/or treated with RNAi against autophagy genes or vector control are presented. Worms were incubated at 20°C and transferred every other day to fresh control L4440 or RNAi plates containing 10 μM AA, 10 μM DGLA, 20 μM EPA, or an equal volume of carrier until cessation of reproduction. Worms were then transferred every 7 d to maintain the strength of the treatments. Statistical analyses of independent experiments are presented in Supplemental Table S2B.

Figure 5.

Lipolysis products activate autophagy in C. elegans distal from their production site with the contribution of LBPs. (A) C. elegans genes encoding the lipid transporter LBPs are transcriptionally up-regulated upon fasting. lbp-1 to lbp-9 mRNA levels of expression in 6-h-fasted wild-type young adults relative to ad libitum-fed animals of the same developmental age are depicted as mean ± SEM of three independent experiments. (B) LIPL-4Ox is sufficient to transcriptionally activate the fasting-responsive lipid transporters lbp-3, lbp-5, and lbp-8. lbp-1 to lbp-9 mRNA levels of expression in LIPL-4Ox relative to control (nontransgenic siblings) incubated at 20°C in OP50 plates are presented as mean ± SEM of three independent experiments. (C) LBP-3 and LBP-5 facilitate activation of autophagy distal from the LIPL-4 site of expression. Representative images of LGG-1∷GFP signal in intestinal and hypodermal seam cells (yellow arrow) of L3–L4 animals overexpressing LIPL-4 only in the digestive tract (vha-6 promoter), treated or not from the L4 stage of the first generation with RNAi against lbp-3 and lbp-5 (50% each), are presented. (D) LBP-3 and LBP-5 contribute to the LIPL-4 overexpression extended life span phenotype. Survival curve of LIPL-4Ox or control (nontransgenic siblings) incubated at 20°C in OP50 plates and transferred as L4 larvae to control or lbp-3/lbp-5 RNAi plates is presented. Worms were incubated at 20°C and transferred every other day to fresh control L4440 or RNAi plates until cessation of reproduction. Worms were then transferred every 7 d to maintain the strength of the treatments. Detailed statistical analysis of an independent experiment is presented in Supplemental Table S3.