Error bars indicate standard error of the mean (SEM), calculated from at least three biological replicates, unless otherwise indicated. P-values were calculated using one-way ANOVA with Tukey’s test or log-rank test for lifespan, unless otherwise indicated and reported as P-values. All statistical tests were performed using GraphPad Prism 7 software.

All strains were grown at 20°C using standard C. elegans methods as previously described (Koh et al., 2018). Nematode growth media (NGM) agar plates were seeded with Escherichia coli strain OP50 for normal growth. C. elegans strains wild type N2, fat-1(bx24), mev-1(tk22), gst-4p::GFP::NLS(cl2166), sod-3p::GFP(cf1553) and bacteria strains OP50 were gifted from the Caenorhaditis Genetics Center. Trilinolenin [TG(54:9)] was obtained from Nu-Chek Prep and deuterated at bis-allylic position as previously described (Smarun et al., 2017) to obtained a mixture of 76% deuterated TG(54:9) [D-TG(54:9)]. Lipids were stored into an atmosphere of argon to prevent lipid oxidation. Lipids were freshly dissolved in PBS buffer (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4) containing 0.1% Triton X-100 or kept at -80°C for later use prior to supplementing NGM agar plate as previously described (Deline et al., 2013). Butylated hydroxytoluene (BHT) and paraquat (PQ) were obtained from Sigma and Acros Organics, respectively.

Lifespan assays were performed at 20°C as previously described (Apfeld and Kenyon, 1999). Synchronized animals were transferred to NGM plates containing 0.2 mM BHT, 1 mM paraquat (PQ), 0.48 mM TG(54:9), or 0.48 mM D-TG(54:9), when indicated. Pyrimidine analog 5-fluoro-2’-deoxyuridine (FUdR, Sigma; St. Louis) was added at 50 μM to pre-fertile young adult worms to prevent development of progeny. Adults were scored manually as dead or alive every 2–3 day. Nematodes which ceased pharyngeal pumping and had no respond to gentle stimulation were recorded as dead. Those worms that were male, crawled off the plate or non-natural death were censored.

Synchronized L1 animals were transferred to NGM plates containing 0.48 mM TG(54:9), 0.48 mM D-TG(54:9) or carrier. Approximately 10,000 L4 to young adult worms were harvested and washed thoroughly with M9 buffer and lyophilised overnight (Vertis, Warminster, PA, United States). FAs were esterified to fatty acid methyl esters (FAME) with 300 μl of 1.25 M HCl-methanol for 1 h at 80°C. FAMEs were extracted three times with 1 ml of hexane. Combined extracts were dried under nitrogen, resuspended in 100 μl hexane. FAMEs were separated by gas chromatography with flame ionization detector (GC-FID; GC-2014; Shimadzu, Kyoto, Japan) using an ULBON HR-SS-10 50 m × 0.25 mm column (Shinwa, Tokyo, Japan). Supelco 37 component FAME mix was used to identify corresponding FAs (Sigma-Aldrich, St. Louis, MO, United States). Data was normalized using internal standard pentadecanoic acid (C15:0) and worm dry weight.

Synchronized L1 mev-1 mutants were transferred to NGM plates containing 0.1 mM BHT, 0.48 mM TG(54:9), 0.48 mM D-TG(54:9) or carrier for 54 h, after which they were transferred on supplemented plates with 8 mM paraquat when indicated for 24 h. Approximately 1,000 L4 to adult worms were harvested and washed thoroughly with M9 buffer, resuspended in 300 μl RIPA buffer (50 mM Tris–HCL pH7.5, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS), lysed with 1 mm silica beads by bead beating. Protein concentration was carried out using the Bicinchoninic Acid (BCA) Protein Assays kit following manufacturer’s protocol (Sigma; St. Louis). Assays for lipid peroxidation, using the thiobarbituric acid reactive substrate (TBARS) kit were performed following manufacturer’s protocol (Cayman Chemical).

Synchronized L1 mev-1 animals were transferred to NGM plates containing 0.1 mM BHT, 0.48 mM TG(54:9), 0.48 mM D-TG(54:9) or carrier and supplemented with 5 mM paraquat when indicated. L4 to young adult worms were transferred to 10 μM BODIPY 581/591 undecanoic acid (BODIPY^581-591-C11) solution and stained for 30 min and washed three times with M9. Images were captured using confocal fluorescence microscope Zeiss LSM 710 microscope with a 20 × objective (Carl Zeiss MicroImaging). Oxidized and non-oxidized BODIPY^581-591-C11 were excited at 488 and 568 and images were collected from emission at 530(30) and 590(30) nm, respectively. Fluorescence signal ratio of oxidized to non-oxidized BODIPY was normalized to carrier.

Synchronized L1 gst-4p::GFP or sod-3p::GFP worms were transferred to NGM plates containing 0.48 mM TG(54:9), 0.48 mM D-TG(54:9) or carrier and supplemented with 5 mM paraquat when indicated. To quantify GFP signal, worms were immobilized with 25 mM tetramisole and mounted on 2% agarose pad. Images were captured using Zeiss Axiovert 200 M fluorescence microscope with a 20 × objective. Images were stitched, and total fluorescence were quantified using Fiji ImageJ software.

Synchronized L1 WT and fat-1(lof) animals were transferred to NGM plates containing 0.48 mM TG(54:9), or 0.48 mM D-TG(54:9) and supplemented with 5 μM 5-fluoro-2’-deoxyuridine (FUdR, Sigma; St. Louis) to facilitate the counting of the eggs. The fertility of the worms was scored by counting eggs on day 5 and 6 after the transfer of the adults to a new plate.

In human, FAs lineloic acid (LA; omega-6) and α-linolenic acid (ALA; omega-3) are essentials (MacLean et al., 2004). In contrast, C. elegans synthesize both LA and ALA consequently we selected omega-3 fatty acid desaturase fat-1(loss-of-function; lof) mutant animal to mimic the human dietary requirement of ALA (Watts and Browse, 2002; Figure 1A). We tested different ROS agents including paraquat (PQ) and 95% oxygen (data not shown). Paraquat is reduced into radical from the mitochondrial leaking electrons to induce the production of superoxide radicals such as superoxide anion radical (O₂^∙-). In turn, hydroperoxyl radicals HO₂^∙ modify PUFAs through oxidative damage in a chain-propagation fashion (Bielski et al., 1983; De Grey, 2002). As low levels of oxidative stress has been reported to increase the lifespan of C. elegans (Wei and Kenyon, 2016) while high levels decrease lifespan (Schaar et al., 2015), we carried out a lifespan assay with fat-1(lof) worms. As expected, high concentration of 1 mM paraquat significantly reduced the lifespan of fat-1(lof) worms compared to carrier (Figure 1B and Table 1). This result indicates that fat-1(lof) worms are sensitive to the production of ROS by the toxic level of paraquat. We also subjected fat-1(lof) worms to the antioxidant BHT but the lifespan was unchanged compared to carrier. This result suggests that fat-1(lof) basal level of ROS is not harmful and neutralising ROS further with antioxidant does not influence fat-1(lof) longevity.

The protective effect of D-PUFAs against ROS has been demonstrated in yeast (Hill et al., 2011; Cotticelli et al., 2013; Andreyev et al., 2015), in primary co-cultures of neurons and astrocytes (Angelova et al., 2015) as well as in AD mouse model Aldh2^-/- (Elharram et al., 2017) by supplementing the media with deuterated LA or ALA (Figure 1C; Hill et al., 2011; Cotticelli et al., 2013; Andreyev et al., 2015). We generated deuterated ALA from trilinolenin [TG(54:9)] to obtain deuterated trilinolenin [D-TG(54:9)] as triglycerides reflect human intake mainly consisting of esterified FAs (Figure 1D). The replacement of hydrogen by deuterium at the potential four bis-allylic CH₂ group between the two double bonds was effective at 97.5% (data not shown) using our recently reported site-specific deuteration of polyunsaturated alkenes method (Smarun et al., 2017). To ensure that C. elegans ingests and metabolizes D-TG(54:9) as well as non-deuterated TG(54:9), we fed L1 larvae fat-1(lof) mutant with standard diet on NGM plates supplemented with 0.48 mM TG(54:9), D-TG(54:9), or the carrier (0.1% Triton X-100 in PBS). C. elegans is typically fed E. coli OP50 bacteria in which PUFAs are naturally absent but can be easily incorporated if supplemented to the media (Webster et al., 2013). Worms were harvested at stage larva 4 (L4)/young adult and lyophilized. Dried worms were subjected to derivatisation with hydrogen chloride in methanol to generate FAME. FAMEs were extracted with hexane and separated on GC-FID using a capillary column (Ulbon HR-SS-10). We observed a significant accumulation of eicosatetraenoic acid (ETA) and eicosapentaenoic acid (EPA) in fat-1(lof) fed D-TG(54:9) while the other changes were statistically non-significant compared to carrier (Figure 1E). This result indicates that deuterated α-linolenic acid is metabolized by C. elegans as a precursor to synthesize PUFAs with higher degrees of unsaturation.

Next, we asked if D-TG(54:9) is toxic to fat-1(lof) mutant by measuring its fertility which is diminished by ROS (Alcantar-Fernandez et al., 2018). As Δ9 fatty acid desaturases fat-6;fat-7 double mutant exhibits lower fertility (Brock et al., 2007), we monitored the number of laid eggs in wild-type (WT, N2) and fat-1(lof) mutant with standard diet on NGM plates supplemented with 0.48 mM TG(54:9), D-TG(54:9), or the carrier. On carrier, fat-1(lof) mutant laid a significant lower amount of eggs compared to WT (Figure 1F). Similarly, fat-1(lof) mutant, fed TG(54:9), laid a similar amount of eggs per worm compared to fat-1(lof) mutant on carrier. To our surprise, fat-1(lof) mutant, fed D-TG(54:9), laid a similar amount of eggs per worm compared to WT. This result suggests that D-TG(54:9) promotes fertility in fat-1(lof) mutant while TG(54:9) might promote lipid peroxidation.

As PUFAs contribute to the generation of oxidative stress through the propagation of lipid peroxide (Porter et al., 1995; Yin et al., 2011; Figure 2A), we hypothesized that TG(54:9) will intensify paraquat-induced lipid peroxidation. Lipid peroxidation was monitored by measuring thiobarbituric acid reactive substances (TBARS) (Lagman et al., 2015) in lipid peroxidation-sensitive mev-1(lof) mutant worms (Labuschagne et al., 2013). The protein MEV-1 is the homolog of succinate dehydrogenase cytochrome b560 subunit of the mitochondrial respiratory chain complex II (Ishii et al., 1998). Synchronized L1 mev-1(lof) mutants were grown on NGM plates seeded with bacteria supplemented with carrier, BHT, TG(54:9), or D-TG(54:9). Subsequently, L4/young adult mev-1(lof) mutants were exposed to 24 h of paraquat. Paraquat alone was insufficient to significantly increase the accumulation of lipid peroxide compared to carrier alone (Figure 2B). On the other hand, mev-1(lof) mutants grown on TG(54:9) and subjected to paraquat exhibited a dramatic increase in lipid peroxides compared to paraquat alone and to TG(54:9) in the absence of paraquat. This result reinforces the role of PUFAs in catalyzing the propagation of ROS through lipid peroxidation (Yang et al., 2016). D-TG(54:9) was sufficient to prevent the propagation of paraquat-induced ROS through lipid peroxidation. This result indicates that D-PUFAs, in the form of triacylglycerol, are resistant to ROS and thus preventing further cellular damages.

To further assess the role of D-TG(54:9) in preventing the formation of lipid peroxides, we monitored in vivo fluorescence of the sensor BODIPY^581/591 undecanoic acid (BODIPY^581/591-C11). The sensor emission peak shifts from 590 to 510 nm when the polyunsaturated butadienyl domain is oxidized (Naguib, 1998; Aldini et al., 2001). mev-1(lof) mutants were grown and treated as for the TBARS assay. In contrast to the TBARS assay, paraquat alone was sufficient to significantly induce lipid peroxidation (Figure 2C,D). This suggests that BODIPY^581/591-C11 is more sensitive than the TBARS assay to monitor lipid peroxidation in C. elegans as previously reported in different cell types (Dominguez-Rebolledo et al., 2010). In contrast to the TBARS assay, TG(54:9) combined with paraquat did not induce lipid peroxidation further compared to paraquat alone in mev-1(lof) mutants. D-TG(54:9) was sufficient to protect mev-1(lof) mutants against paraquat-induced lipid peroxidation. As the BODIPY-C11 lipid peroxidation reporter assay yielded small differences, BODIPY possibly failed to be incorporated evenly through the animal and perhaps not sufficiently where most of lipid peroxidation occurs. Together with the TBARS assay, these results demonstrate that D-PUFA is sufficient to prevent lipid peroxidation in C. elegans.

To further assess the role of D-TG(54:9) in preventing the propagation of lipid peroxide, we monitored the oxidative stress response in vivo. We monitored the expression of the reporter protein GFP under the promotor sod-3 (sod-3p::GFP) (Xu and Kim, 2012). SOD-3 is a mitochondrial superoxide dismutase which has been reported to increase transcriptionally as a result of oxidative stress (An and Blackwell, 2003). These worms were grown and treated as per the TBARS assay. No significant increase in the expression of sod-3p::GFP was observed across the different conditions (Figure 3A,B). As this assay was inconclusive, we used the transgenic worm expressing GFP tagged with a nuclear localisation signal under the promotor gst-4 (gst-4p::GFP::NLS) (Paek et al., 2012). GST-4 is a glutathione S-transferase protein that responds to oxidative stress by an increase at the transcription and translational levels (Link and Johnson, 2002). Glutathione S-transferases protect cells against lipid peroxidation (Yang et al., 2001). gst-4p::GFP::NLS worms were grown with triacylglycerol and paraquat as per the TBARS assay. The expression of gst-4p::GFP::NLS was significantly increased in the presence of paraquat and further increase with the addition of TG(54:9) (Figures 3C,D). On the other hand, no significant change in GFP expression was observed in animals fed D-TG(54:9) in the absence or the presence of paraquat. Consistent with the TBARS assay, these findings further demonstrate that TG(54:9) is harmful by exponentially propagating paraquat-induced ROS while D-TG(54:9) exhibit a strong protective effect.

As D-TG(54:9) reduces oxidative stress, we carried out a lifespan assay of fat-1(lof) worms fed normal diet supplemented with either TG(54:9) or D-TG(54:9). D-TG(54:9) significantly extended the lifespan of worms compared to the supplementation with TG(54:9) (Figure 4A and Table 1). This suggests that D-TG(54:9) might be sufficient to prevent endogenous ROS-induced cellular damages associated with aging. To further assess the role of D-TG(54:9) on the lifespan of fat-1(lof) worms, we exposed the animals to paraquat from L1 stage. As expected, the lifespan of fat-1(lof) worms exposed to paraquat was not significantly extended by TG(54:9) (Figure 4B and Table 1). As fat-1(lof) worms are unable to synthesize several PUFAs (Figure 1A), TG(54:9) might be necessary as a precursor of PUFAs ETA and EPA to extend the lifespan although the animals are under oxidative stress condition. It should be noted that worms were exposed to a fifth of the paraquat concentration used to monitor lipid peroxidation and oxidative stress. The lifespan of fat-1(lof) worms exposed to paraquat was further extended with D-TG(54:9) compared to paraquat alone and to TG(54:9) in combination with paraquat. This result indicates that D-TG(54:9) is sufficient to promote longevity. Potentially, supplementing a human diet with D-PUFAs might be sufficient to decelerate aging and to prevent the progression of age-associated diseases.