The ω-3 fatty acid α-linolenic acid extends Caenorhabditis elegans lifespan via NHR-49/PPARα and oxidation to oxylipins

Abstract

The dietary intake of ω-3 polyunsaturated fatty acids has been linked to a reduction in the incidence of aging-associated disease including cardiovascular disease and stroke. Additionally, long-lived Caenorhabditis elegans glp-1 germ line-less mutant animals show a number of changes in lipid metabolism including the increased production of the ω-3 fatty acid, α-linolenic acid (ALA). Here, we show that the treatment of C. elegans with ALA produces a dose-dependent increase in lifespan. The increased longevity of the glp-1 mutant animals is known to be dependent on both the NHR-49/PPARα and SKN-1/Nrf2 transcription factors, although the mechanisms involved are incompletely understood. We find that ALA treatment increased the lifespan of wild-type worms and that these effects required both of these transcription factors. Specifically, NHR-49 was activated by ALA to promote the expression of genes involved in the β-oxidation of lipids, whereas SKN-1 is not directly activated by ALA, but instead, the exposure of ALA to air results in the oxidation of ALA to a group of compounds termed oxylipins. At least one of the oxylipins activates SKN-1 and enhances the increased longevity resulting from ALA treatment. The results show that ω-3 fatty acids inhibit aging and that these effects could reflect the combined effects of the ω-3 fatty acid and the oxylipin metabolites. The benefits of ω-3 fatty acid consumption on human health may similarly involve the production of oxylipins, and differences in oxylipin conversion could account for at least part of the variability found between observational vs. interventional clinical trials.

Keywords: Caenorhabditis elegans; NHR-49; SKN-1; aging; oxylipin; ω-3 fatty acids.

Figure 1

α‐linolenic acid (ALA) treatment increases worm lifespan via nhr‐49/PPARα. (A) Structure of ALA showing the locations of the desaturated carbon bonds in the fatty acid backbone. (B) Treatment of spe‐9; fer‐15 worms with ALA at concentrations of 2–10 mm increases lifespan, compared to control animals treated with the ALA solvent alone, with the maximal effect being observed at the 5 mm dose. N > 80 for all treatments. P < 0.0001 for control vs. 5 mm ALA treatment by log‐rank test. (C) Treatment of wild‐type N2 worm with 5 mm ALA increases lifespan, compared to control animals treated with the ALA solvent alone. N > 75 for both treatments. P < 0.0001 by log‐rank test. The expression of an acs‐2p::GFP reporter is increased after treatment of 5 mm ALA as shown by fluorescence microscopy (D) or quantitation of the GFP fluorescence in the images using the ImageJ program (F). The increase in expression requires nhr‐49/PPARα as the increase in expression is blocked by nhr‐49 RNAi. N > 6 for all RNAi and ALA treatment combinations. *** represents P < 0.001 for Control RNAi ‐ ALA vs. Control RNAi +ALA and Control RNAi +ALA vs. nhr‐49 RNAi + ALA by t‐test. (E) Inhibition of acs‐2 and acs‐7 in spe‐9; fer‐15 worms by RNAi reduces the effects of ALA treatment on lifespan. N > 100 for all treatments. P = 0.0006 for acs‐2 + acs‐7 RNAi ALA treated vs. control RNAi ALA treated by log‐rank test. P = NS for acs‐2 + acs‐7 RNAi control treated vs. control RNAi control treated by log‐rank test. P < 0.0001 for control RNAi control treated vs. control RNAi ALA treated. (G) The increase in worm lifespan produced by treatment with 5 mm ALA requires nhr‐49 because spe‐9; fer‐15 worms treated with nhr‐49 RNAi show no increase in lifespan following ALA treatment. N > 72 for all treatments. P < 0.0001 for control RNAi −ALA vs. +ALA and P = NS for nhr‐49 RNAi −ALA vs. +ALA.

Figure 2

α‐linolenic acid (ALA) treatment activates the skn‐1/Nrf2 transcription factor. Treatment of worms carrying a gst‐4p::GFP transgene with 5 mm ALA leads to an increase in GFP expression as shown by fluorescence microscopy (A) or quantitation of the GFP fluorescence in the images using the ImageJ program (B). The increased GFP expression produced by ALA treatment requires both nhr‐49 and skn‐1 as either RNAi treatment blocks the increase. N > 6 for all RNAi and ALA treatment combinations. *** represents P < 0.001 for control RNAi –ALA vs. Control RNAi +ALA, nhr‐49 RNAi +ALA vs. Control RNAi +ALA, and skn‐1 RNAi +ALA vs. Control RNAi +ALA. The measurement of gene expression via Nanostring also shows an increase in gst‐4 expression (C) as well as the oxidative stress response genes gcs‐1 (D), gst‐7 (E), and nit‐1 (F). However, only the increased expression of gst‐4 (C) is inhibited by nhr‐49 RNAi whereas increases in expression for gcs‐1 (D), gst‐7 (E), and nit‐1 (F) still occur. In contrast, skn‐1 RNAi treatment blocks the increased expression of all of these genes. N = 4 for all Nanostring experiments. *** represents P < 0.001, ** represents P < 0.01 and + represents 0.05 < P < 0.1.

Figure 3

Control of gst‐4 expression by α‐linolenic acid (ALA) and oxidative stress. ALA treatment activates the expression of the gst‐4p::GFP reporter as shown by fluorescence microscopy (A top panels) and quantitation of GFP fluorescence (B). N = 6 and *** represents P < 0.001 for ‐NAC ‐ ALA vs. −NAC +ALA. However, the effects of ALA on reporter activation do not involve the production of reactive oxygen species (ROS) because the activation is not blocked by pretreatment with N‐acetylcysteine (NAC) (A bottom panels and B). N = 6 and P = 0.31 ALA treatment +/− NAC. In contrast, NAC treatment is able to attenuate the activation of the gst‐4p::GFP reporter produced by oxidative stress produced by juglone treatment as shown by fluorescence microscopy (C) and quantitation of GFP fluorescence (D). N = 6 for all treatments. *** represents P < 0.0001 by t‐test and ** represents P = 0.0006 by t‐test. (E) After exposure to oxidative stress produced by juglone treatment, both the skn‐1/Nrf2 transcription factor and the nhr‐49/PPARα gene are required for the activation of the gst‐4p::GFP reporter as shown by fluorescence microscopy (E) and quantitation of GFP fluorescence (F). N = 6 and *** represents P < 0.001 for Control RNAi ‐ juglone vs. Control RNAi +juglone, Control RNAi + juglone vs. nhr‐49 RNAi +juglone and Control RNAi + juglone vs. skn‐1 RNAi +juglone.

Figure 4

α‐linolenic acid (ALA) treatment increases worm lifespan via skn‐1/Nrf2. (A) The increase in worm lifespan produced by treatment with 5 mm ALA requires skn‐1 because animals treated with skn‐1 RNAi show no increase in lifespan following ALA treatment. N = 83 for control RNAi ‐ALA, 95 for control RNAi +ALA, 75 for skn‐1 RNAi ‐ALA, 56 for skn‐1 RNAi +ALA. P < 0.0001 for control RNAi ‐ALA vs. +ALA by log‐rank test, and P = NS for skn‐1 RNAi ‐ALA vs. +ALA by log‐rank test. (B) The activation of the acs‐2p::GFP reporter after ALA treatment is not reduced by blocking the synthesis of longer‐chain ω‐3 fatty acids with fat‐3 or elo‐1 RNAi as shown by digital imaging or the quantification of GFP fluorescence (C). N = 6 for all treatments. *** represents P < 0.0001 by t‐test. (D) The activation of the gst‐4p::GFP reporter after ALA treatment is also not blocked by fat‐3 or elo‐1 RNAi treatment as shown by digital imaging or quantification of GFP fluorescence (E). N = 6 for all treatments. *** represents P < 0.0001 by t‐test. (F) The activation of the acs‐2p::GFP and gst‐4p::GFP reporters still occurs when worms are fed ALA with heat‐killed bacteria as shown by digital imaging or the quantification of GFP fluorescence (G). N = 6 for all treatments. *** represents P < 0.0001 by t‐test.

Figure 5

Oxidation of α‐linolenic acid (ALA) activates skn‐1 via the production of oxylipins. The exposure of ALA to either oxygen in room air (B) or to the oxidizer 2,2′‐Azobis(2‐methylpropionamidine) dihydrochloride (AAPH) (C) leads to the accumulation of the oxylipin 9S‐hydroperoxy‐10E,12Z,15Z‐octadecatrienoic acid (9(S)‐HpOTrE) as shown via the use of high‐performance liquid chromatography followed by mass spectrometry compared to a freshly opened aliquot of ALA (A). Shown in (A) is the structure of 9(S)‐HpOTrE as an inset which shows the shift in desaturations and presence of a peroxide group compared to ALA. As a result of the oxidation of the ALA to 9(S)‐HpOTrE, the percentage of each sample that is 9(S)‐HpOTrE compared to ALA also increases from 0.3% to almost 1.8% (D). N = 5 for all samples. ***P < 0.0006 for fresh ALA vs. air‐oxidized ALA. P = 0.0078 for fresh ALA vs. AAPH‐oxidized ALA by t‐test. (E) The treatment of worms with the AAPH‐oxidized ALA leads to a greater increase in the expression of the gst‐4p::GFP reporter (E bottom right) compared to the treatment of worms with either the unoxidized ALA (E bottom left) or the vehicle solution treated with AAPH and then neutralized in a similar fashion (E top right). (F) These effects are also seen when the fluorescence in additional images are measured by the ImageJ program. N = 6 and *** represents P < 0.001 for −AAPH −ALA vs. −AAPH +ALA and −AAPH +ALA vs. +AAPH +ALA. (G) The treatment of worms carrying the gst‐4p::GFP reporter with 9(S)‐HpOTrE but not the isomer 13(S)‐HpOTrE at a concentration of 0.2 mm leads to an increase in GFP fluorescence. (H) Quantification of GFP fluorescence from animals treated with the specified oxylipin and then imaged as in (G). N = 6 and *** represents P < 0.001 for control vs. 9(S)‐HpOTrE treatment.

Figure 6

The oxylipin 9S‐hydroperoxy‐10E,12Z,15Z‐octadecatrienoic acid (9(S)‐HpOTrE) acts via the skn‐1 transcription factor and enhances the effects of α‐linolenic acid (ALA) on worm lifespan. (A) The gst‐4p::GFP reporter is activated by 9(S)‐HpOTrE and requires the nhr‐49 and skn‐1 transcription factors (top panels) whereas 9(S)‐HpOTrE has little effect on the expression of the acs‐2p::GFP reporter (bottom panels). These effects are also seen in larger groups of worms that are similarly treated and imaged with the images being used for quantification of change in GFP fluorescence for the gst‐4p::GFP (B) and acs‐2p::GFP (C) reporters. For (B), N = 6 for all treatments. *** represents P < 0.0001 by t‐test. For (C), N = 6 for all treatments. P = NS for control vs. 9(S)‐HpOTrE treatment of the acs‐2p::GFP reporter. (D) The supplementation of a freshly opened ALA stock does not affect the expression of the lipid metabolism gene acs‐2 (A left panels) but produces a further increase in the expression of gst‐4 (right panels). These effects are also seen in larger groups of worms that are similarly treated and imaged with the images being used for quantitation of changes in GFP fluorescence for the acs‐2p::GFP (E) and gst‐4p::GFP reporters (F). For (E), N = 6 for each treatment and *** represents P < 0.001 for control vs. ALA. P = 0.45 for ALA vs. ALA + 9(S)‐HpOTrE. For (F), N = 6 for each treatment and *** represents P < 0.001 for control vs. ALA and ALA vs. ALA + 9(S)‐HpOTrE. (G) Treatment of worms with ALA‐supplemented 9(S)‐HpOTrE leads to a greater increase in worm lifespan compared to the treatment of worms with freshly prepared ALA alone N = 58 for control treatment, 39 for ALA alone, and 50 for ALA + 9(S)‐HpOTrE. P = 0.0013 for control vs. ALA alone by log‐rank test, and P = 0.04 for ALA vs. ALA + 9(S)‐HpOTrE by log‐rank test.