Lipid droplets and peroxisomes are co-regulated to drive lifespan extension in response to mono-unsaturated fatty acids

Abstract

Dietary mono-unsaturated fatty acids (MUFAs) are linked to longevity in several species. But the mechanisms by which MUFAs extend lifespan remain unclear. Here we show that an organelle network involving lipid droplets and peroxisomes is critical for MUFA-induced longevity in Caenorhabditis elegans. MUFAs upregulate the number of lipid droplets in fat storage tissues. Increased lipid droplet number is necessary for MUFA-induced longevity and predicts remaining lifespan. Lipidomics datasets reveal that MUFAs also modify the ratio of membrane lipids and ether lipids-a signature associated with decreased lipid oxidation. In agreement with this, MUFAs decrease lipid oxidation in middle-aged individuals. Intriguingly, MUFAs upregulate not only lipid droplet number but also peroxisome number. A targeted screen identifies genes involved in the co-regulation of lipid droplets and peroxisomes, and reveals that induction of both organelles is optimal for longevity. Our study uncovers an organelle network involved in lipid homeostasis and lifespan regulation, opening new avenues for interventions to delay aging.

Fig. 1. MUFAs upregulate the number of lipid droplets in the intestine.

a, Schematic of genetic and dietary interventions that lead to MUFA accumulation in C. elegans. Mammalian gene names are indicated first. In all figures and panels, worms are hermaphrodites (female-like), except for Fig. 1g and Extended Data Fig. 1g, where males are used. b,c, Intestinal puncta, assessed by SRS microscopy, following MUFA accumulation. b, SRS image of total lipids in the anterior part of one worm (head and intestine; top). Zoomed-in images of the intestine (bottom). Scale bars, 100 µm (top) and 5 µm (bottom). c, Number of intestinal puncta in n = 18, 30, 27 and 19 worms treated with control, fat-7, ash-2 and fat-2 RNAi, respectively. Puncta intensities are provided in Extended Data Fig. 1e. d, Intestinal lipid droplets, assessed by SRS microscopy, in worms expressing the lipid droplet protein DHS-3 fused to GFP driven by the endogenous dhs-3 promoter (intestinal expression; dhs-3p::dhs-3::GFP) following MUFA accumulation. Zoomed-in images of the intestine. Scale bar, 5 µm. Lipid droplet numbers, as assessed by double-positive puncta, are provided in Extended Data Fig. 1f. e,f, Number of intestinal lipid droplets, assessed by fluorescence, in dhs-3p::dhs-3::GFP worms following MUFA accumulation. e, Fluorescence image of the anterior part of one worm (head and intestine; top). Zoomed-in fluorescence images of the intestine (bottom). Scale bars, 100 µm (top) and 5 µm (bottom). f, Number of lipid droplets in n = 17, 12, 17 and 13 worms treated with control, fat-7, ash-2 and fat-2 RNAi, respectively. g, Number of intestinal lipid droplets, assessed by fluorescence, in dhs-3p::dhs-3::GFP worms (n = 22 male worms for each condition) following MUFA accumulation. h, Hypodermal lipid droplet number—assessed by fluorescence in worms expressing the lipid droplet protein PLIN-1 fused to mCherry driven by the endogenous plin-1 promoter (ubiquitous expression; plin-1p::plin-1::mCherry)—following MUFA accumulation. Zoomed-in images of the hypodermis. Scale bar, 5 µm. Lipid droplet numbers are provided in Extended Data Fig. 1h. Elements of a, d and h are created with BioRender.com. c,f,g, Data are representative of three (c,f) or two (g) independent experiments. Each dot represents the number of puncta in a 26 × 26 µm² area in the intestine of an individual worm. Data are the mean ± s.d. P values were determined using a two-tailed Mann–Whitney test. Source data are provided. Source data

Fig. 2. Supplementation with cis-MUFA (oleic acid) but not trans-MUFA (elaidic acid) increases lipid droplet number and extends lifespan.

a, Chemical structure of the cis-MUFA oleic acid and the trans-MUFA elaidic acid. b,c, Number of intestinal lipid droplets, assessed by fluorescence, in dhs-3p::dhs-3::GFP worms following supplementation with sterically different dietary MUFAs. b, Zoomed-in images of the intestine. Scale bar, 5 µm. c, Number of lipid droplets in n = 31, 29 and 28 worms following control, dietary oleic acid and dietary elaidic acid supplementation, respectively. Data are the mean ± s.d. Each dot represents the number of puncta in a 26 × 26 µm² area in the intestine of an individual worm. P values were determined using a two-tailed Mann–Whitney test. d, Cis-MUFA (oleic acid), but not trans-MUFA (elaidic acid), extends lifespan; n ≥ 128 worms for each condition. Percentages of the median lifespan extension and P values (log-rank Mantel–Cox test) are indicated; NS, not significant. c,d, Data are representative of three (d) or four (c) independent experiments. Source data are provided. Source data

Fig. 3. Increased lipid droplet number is necessary for MUFA-induced longevity and sufficient to extend lifespan.

a, Schematic of conserved proteins involved in lipid droplet synthesis and degradation. Mammalian protein names are indicated first. Created with BioRender.com. b,c, Number of intestinal lipid droplets, measured by fluorescence, in dhs-3p::dhs-3::GFP worms following lpin-1 depletion and MUFA accumulation. b, Number of lipid droplets in n = 16, 20, 23 and 19 worms treated with control, ash-2, lpin-1, and ash-2 + lpin-1 RNAi, respectively (Extended Data Fig. 2c for the efficiency of the double knockdown). c, Number of lipid droplets in n = 35, 29, 35 and 32 worms treated with control or dietary oleic acid supplementation in the absence or presence of lpin-1 RNAi, respectively. d, lpin-1 is necessary for longevity following ash-2 depletion; n ≥ 96 worms for each condition. e, lpin-1 is necessary for longevity following dietary supplementation with oleic acid; n ≥ 105 worms for each condition. f, Number of intestinal lipid droplets, assessed by fluorescence, in dhs-3p::dhs-3::GFP worms following seip-1 and ash-2 depletion. Zoomed-in images of the intestine. Scale bar, 5 µm. Lipid droplet numbers are provided in Extended Data Fig. 2e. g, seip-1(gk5008) is necessary for longevity following ash-2 depletion; n ≥ 64 worms for each condition. h, seip-1(gk5008) is necessary for longevity following fat-2 depletion; n ≥ 90 worms for each condition. i, Number of intestinal lipid droplets, measured by fluorescence, in dhs-3p::dhs-3::GFP worms following hosl-1 depletion; n = 28 and 25 worms treated with control and hosl-1 RNAi, respectively. b,c,i, Each dot represents the number of lipid droplets in a 26 × 26 µm² area of the intestine of an individual worm. Data are the mean ± s.d. P values were determined using a two-tailed Mann–Whitney test. j, hosl-1 depletion is sufficient to extend lifespan; n ≥ 94 worms for each condition. d,e,g,h,j, Percentages of median lifespan extension and P values are indicated. P values were determined using a log-rank Mantel–Cox test. b–e,g–j, Data are representative of three (b–e,g,i,j) or two (h) independent experiments. NS, not significant; WT, wild-type worms. Source data are provided. Source data

Fig. 4. Increased numbers of lipid droplets in young or middle-aged individuals is predictive of a long life.

a, Experimental set-up for sorting worms according to the fluorescence intensity of the DHS-3::GFP lipid droplet reporter in dhs-3p::dhs-3::GFP worms using a large-particle BioSorter. Created with BioRender.com. b,c, Number of intestinal lipid droplets, assessed by fluorescence, in a synchronized population of young adult (adult day 1) dhs-3p::dhs-3::GFP worms after sorting using a BioSorter. b, Zoomed-in images of the intestine. Scale bar, 5 µm. c, Number of lipid droplets in n = 15 worms for each condition. d, Worms sorted at young-adult age (adult day 1) with high numbers of lipid droplets live longer than worms with low numbers of lipid droplets; n ≥ 117 worms for each condition. e, Number of intestinal lipid droplets, measured by fluorescence, in a synchronized population of middle-aged (adult day 6) dhs-3p::dhs-3::GFP worms after manual sorting; n = 30 worms for each condition. Zoomed-in images are provided in Extended Data Fig. 3b. c,e, Each dot represents the number of lipid droplets in a 26 × 26 µm² area of the intestine of an individual worm. Data are the mean ± s.d. P values were determined using a two-tailed Mann–Whitney test. f, Worms sorted at middle age (adult day 6) with high numbers of lipid droplets live longer than worms with low numbers of lipid droplets; n ≥ 195 for each condition. d,f, Percentages of the median lifespan extension and P values, determined using a log-rank Mantel–Cox test, are indicated. c–f, Data are representative of two (c,d) or three (e,f) independent experiments. Source data are provided. Source data

Fig. 5. Lipidomic datasets of MUFA-enriched worms reveal changes in membrane lipids and predict decreased lipid oxidation.

a, Untargeted lipidomic analysis on whole worms using LC–MS/MS. b, Principal component (PC) analysis of the lipidome separates MUFA-enriched conditions (ash-2 RNAi) from control conditions. c, Fatty acyl chain abundance of saturated fatty acids (SFAs), MUFAs and PUFAs among all lipids in middle-aged worms following ash-2 depletion. d, Lipidomic analysis of MUFAs and PUFAs in membrane lipids (phospholipids) following ash-2 depletion. e, Lipidomics analysis of ether lipids following ash-2 RNAi depletion. f, Schematic indicating that membrane lipid oxidation is generally increased in the presence of PUFA-containing phospholipids and ether lipids, particularly those present in PUFAs. Accumulation of lipid oxidation can lead to membrane damage and loss of membrane integrity. g, Lipidomic analysis of the peroxidation index (probability of lipid oxidation; calculation in Methods) following ash-2 depletion. a and f are created with BioRender.com. b–e,g, Each dot represents a biological replicate; n = 6 independent biological replicates examined in one experiment. c–e,g, Box-and-whisker plots with the median (central line), 25th and 75th percentiles (outer lines), and minimum and maximum within 1.5× the interquartile range (whiskers) indicated. P values were determined using a two-tailed Wilcoxon test with Benjamini–Hochberg test for multiple hypothesis correction. Source data are provided. Source data

Fig. 6. MUFAs decrease age-dependent lipid oxidation and preserve cell and membrane integrity.

a, Relative MDA levels, used as a measure of the level of lipid oxidation, during aging and following ash-2 depletion. Data for n = 4, 13 and 11 samples from control young, control middle-aged (control RNAi) and middle-aged worms treated with ash-2 RNAi, respectively, were normalized to the middle-aged control of the corresponding experiment. b, Lipid oxidation assessed via western blotting for 4-HNE levels during aging and following salinazid treatment. The arrows indicate bands that change following salinazid treatment. c, Levels of lipid oxidation, quantified via MDA levels, following supplementation with oleic acid or elaidic acid; n = 10, 9 and 6 samples from middle-aged worms with control, oleic acid and elaidic acid supplementation, respectively. d,e, Cell and membrane integrity, assessed according to the intensity of propidium iodide (PI) staining, during aging and following ash-2 depletion. d, Images of one worm per condition stained with PI. Dashed lines outline the worms. Scale bar, 100 µm. e, Intensity of PI staining for n = 32, 40, 40 and 55 young and old worms treated with control RNAi, old worms treated with ash-2 RNAi and dead worms, respectively. Each dot represents the mean PI signal in one worm. f, Levels of lipid oxidation, quantified via MDA levels, following oleic acid supplementation and lpin-1 depletion; n = 10, 9, 8 and 8 samples from middle-aged worms with control or oleic acid supplementation in the absence or presence of lpin-1 RNAi, respectively. g, Levels of lipid oxidation, quantified via MDA levels, in seip-1 mutant worms; n = 8 samples from middle-aged worms for each condition. h, Levels of lipid oxidation, quantified via MDA levels, following salinazid treatment; n = 9 samples from middle-aged worms for each condition. a,c,f–h, Each dot represents a biological replicate. Each shape represents an independent experiment. a,c,f–h, Data are the mean ± s.d. of four (a), two (c,f,g) or three (h) independent experiments. P values were determined using a two-tailed Mann–Whitney test. i, Salinazid and ash-2 depletion act in the same pathway to extend longevity. Percentages of the median lifespan extension and P values, determined using a log-rank Mantel–Cox test, are indicated; n ≥ 110 worms for each condition. b,e,i, Data are representative of two (b) or three (e,i) independent experiments. Source data are provided. Source data

Fig. 7. MUFAs upregulate peroxisome number, which is required for MUFA-induced longevity.

a, Organelles such as mitochondria and peroxisomes are also involved in lipid metabolism. Created with BioRender.com. b, Analysis of existing transcriptomic datasets of worms following MUFA accumulation. Upregulated GO terms that are shared between worms treated with either control or ash-2 RNAi. GO terms analyzed using WormEnrichR. GO terms upregulated in middle-aged individuals (adult day 5, whole worms; left column). GO terms upregulated in young individuals (adult day 1/intestine; right column). GO terms were considered significant if they had a combined score of >5 for the log-transformed P value (Fisher’s exact test) multiplied by the rank-based enrichment z-score. ROS, reactive oxygen species. c–e, Number of intestinal peroxisomes, assessed by fluorescence, following MUFA accumulation in worms expressing a peroxisome-localized GFP (GFP–SKL) driven by the intestinal ges-1 promoter (ges-1p::GFP–SKL). c, Zoomed-in images of the intestine. Scale bar, 5 µm. Intensity of peroxisome-localized GFP in Extended Data Fig. 5a. d, Number of peroxisomes following MUFA accumulation in n = 39, 38 and 40 worms treated with control, ash-2 and fat-2 RNAi, respectively. e, Number of peroxisomes following dietary supplementation with oleic acid; n = 40 and 37 control- and oleic acid-supplemented worms, respectively. d,e, Data are the mean ± s.d. Each dot represents the number of peroxisomes in a 26 × 26 µm² area in the intestine of an individual worm. P values were determined using a two-tailed Mann–Whitney test. f, Prx-5 is necessary for longevity following ash-2 depletion. Percentages of the median lifespan extension and P values are indicated; P values were determined using a log-rank Mantel–Cox test; NS, not significant; n ≥ 94 worms for each condition. d–f, Data are representative of three independent experiments. Source data are provided. Source data

Fig. 8. Targeted screen to identify genes involved in the co-regulation of lipid droplet and peroxisome number.

a, Number of intestinal lipid droplets and peroxisomes, measured by fluorescence, in dhs-3p::dhs-3::GFP; vha-6p::mRFP–SKL worms during aging and following ash-2 depletion. Data are the mean ± s.e.m. of n = 21–37 worms. Each dot represents the mean organelle number of all worms imaged for this condition. b, Targeted screen design. Created with BioRender.com c, Genes involved in the co-regulation of organelle number. Number of intestinal lipid droplets and peroxisomes, measured by fluorescence, in dhs-3p::dhs-3::GFP; vha-6p::mRFP–SKL worms following treatment with 62 different RNAis. RNAi of the indicated genes resulted in an increase (red) or decrease (teal) of both organelles; n ≥ 18 worms per condition. d, Number of intestinal lipid droplets and peroxisomes, quantified by fluorescence, in dhs-3p::dhs-3::GFP; vha-6p::mRFP–SKL worms following sbp-1 and nhr-49 transcription factor depletion; n = 24–30 worms. e, Number of intestinal lipid droplets and peroxisomes, quantified as in d, following ash-2, fat-2 or hosl-1 depletion; n = 20–42 worms. f, Genes involved in uncoupling lipid droplet and peroxisome number. RNAi of the indicated genes resulted in decreased numbers of lipid droplets and increased peroxisomes (yellow) or increased numbers of lipid droplets and decreased peroxisomes (blue). g, Number of lipid droplets and peroxisomes, measured as in d, following vps13d depletion; n = 33 or 34 worms. h, Number of lipid droplets and peroxisomes, measured as in d, following rab-7 depletion; n = 29–41 worms. d,e,g,h, Data are the mean ± s.d. Each dot represents the organelle number of an individual worm normalized to control worms. P values were determined using a two-tailed Mann–Whitney test. i, Genes important for lifespan. Perturbations that affect the co-regulation of the number of lipid droplets and peroxisomes, color-coded according to their effect on lifespan and MUFA-induced longevity. c,f,i, Each dot represents the mean organelle number, normalized to control, of all worms imaged for this condition. A two-tailed Pearson’s R² test was used to analyze correlation; dotted line, nonlinear fit. j, vps13d is not necessary for longevity following ash-2 depletion; n ≥ 71 worms for each condition. k, rab-7 is necessary for longevity following ash-2 depletion; n ≥ 93 worms for each condition. j,k, Percentages of the median lifespan extension and P values, determined using a log-rank Mantel–Cox test, are indicated. a,d,e,g,h,j,k, Data are representative of three (d,g,h,j,k) or two (a,e) independent experiments. Source data are provided. Source data

Extended Data Fig. 1. Perturbations that increase MUFAs and their effect on lipid droplets.

a, ash-2 or fat-2 depletion extends lifespan; n ≥ 92 worms for each condition. Percentage of median lifespan extension and P values are indicated. P values: log-rank Mantel–Cox test. b, Dietary oleic acid supplementation extends lifespan; n ≥ 135 worms for each condition. Analysis as in a. c, Fatty acid profile by GC–MS following ash-2 depletion. Fatty acid levels were normalized to the control condition. Data are the mean ± s.d. of three independent experiments, each with three biological replicates. Significant P values are shown. P values: two-way ANOVA with Bonferroni’s multiple comparison test. d, Fatty acid profile by GC–MS following oleic acid supplementation. Analysis as in c. e, Puncta intensity, quantified by SRS microscopy; n = 420, 1,603 and 927 puncta in ≥18 worms treated with control, ash-2 RNAi and fat-2 RNAi, respectively. Data are the mean ± s.d. Each dot represents the SRS signal intensity of one puncta. Lower segment of the y axis displays values of 0–90,000; upper segment of the y axis of 90,000–200,000. P values: two-tailed Mann–Whitney test. f, Quantification of intestinal lipid droplet number (double-positive puncta), measured by SRS microscopy, in dhs-3p::dhs-3::GFP worms following MUFA accumulation; n = 33, 34 and 29 worms treated with control and ash-2 RNAi, and following oleic acid supplementation, respectively. Data are the mean ± s.d. Each dot represents the lipid droplet number in a 26 × 26 µm² area in the intestine of an individual worm. P values: two-tailed Mann–Whitney test. Lipid droplets shown in Fig. 1d. g, Intestinal lipid droplets, assessed by fluorescence, in dhs-3p::dhs-3::GFP worms (males) following ash-2 depletion. Zoomed-in images of the intestine. Scale bar, 5 µm. Lipid droplet number quantified in Fig. 1g. h, Quantification of hypodermal lipid droplet number, measured by fluorescence, in plin-1p::plin-1::mCherry worms following MUFA accumulation; n = 21, 22 and 23 worms treated with control, ash-2 and fat-2 RNAi, respectively. Data are the mean ± s.d. Each dot represents the lipid droplet number in a 15 × 15 µm² area of an individual worm. Lipid droplets shown in Fig. 1h. P values: two-tailed Mann–Whitney test. i,j, Lipid droplets in eggs (in utero), assessed by Nile red fluorescence, following ash-2 depletion. i, Zoomed-in images of one egg per condition. Scale bar, 5 µm. j, Quantification of lipid droplet number; n = 45, 30 and 24 eggs in ≥15 worms treated with control, ash-2 and plin-1 RNAi, respectively. Data are the mean ± s.d. Each dot represents the lipid droplet number in a 16 × 16 µm² area of an individual egg. P values: two-tailed Mann–Whitney test. k,l, Lipid droplet size, measured by fluorescence, in dhs-3p::dhs-3::GFP worms following MUFA accumulation. k, Quantification of lipid droplet diameter; n = 1,720, 875, 1,803 and 1,550 lipid droplets in ≥14 worms treated with control, fat-7, ash-2 and fat-2 RNAi, respectively. Data are the mean ± s.d. Each dot represents the diameter of one lipid droplet. P values: two-tailed Mann–Whitney test. l, Quantification of lipid droplet diameter; n = 1,299 and 2,168 lipid droplets in ≥20 worms following control and oleic acid supplementation, respectively. Analysis as in k. m, Fatty acid profile by GC–MS following elaidic acid supplementation. Data are the mean ± s.d of two independent experiments, each with three biological replicates. Analysis as in c. n, Quantification of intestinal lipid droplet number, measured by fluorescence, in dhs-3p::dhs-3::GFP worms following dietary cis- or trans-vaccenic acid supplementation; n = 24, 26 and 24 worms following control, and dietary cis- and trans-vaccenic acid supplementation, respectively. Analysis as in f. a, Representative of three independent experiments. b, Representative of four independent experiments. e,f,h,j,k,l,n, Representative of two independent experiments. Source numerical data of all experiments, replicates and statistics are provided. Source data

Extended Data Fig. 2. Lipid droplet perturbations that are important for longevity.

a, Endogenous LPIN-1 expression, measured by fluorescence of GFP tagged to the C-terminus of LPIN-1 at the endogenous lpin-1 locus. Fluorescent images of the anterior and posterior parts of young adult lpin-1(wbm76[lpin-1::GFP]) worms. Scale bar, 100 µm. b, Intestinal lipid droplets, assessed by fluorescence, in dhs-3p::dhs-3::GFP transgenic worms following lpin-1 and ash-2 depletion. Zoomed-in images of the intestine. Scale bar, 5 µm. Lipid droplet number is quantified in Fig. 3b. c, RNAi efficiency in conditions using two RNAi constructs. RT–qPCR on RNA extracted from worms treated with control, ash-2, lpin-1 and ash-2 + lpin-1 RNAi. The mRNA levels of target genes relative to act-1 mRNA levels were normalized to the empty vector controls. Data are the mean of two independent experiments, with three replicates each. d, Intestinal lipid droplets, assessed by fluorescence, in dhs-3p::dhs-3::GFP transgenic worms following lpin-1 depletion and oleic acid supplementation. Zoomed-in images of the intestine. Scale bar, 5 µm. Lipid droplet number is quantified in Fig. 3c. e, Quantification of intestinal lipid droplet number, measured by fluorescence, in dhs-3p::dhs-3::GFP worms following seip-1 and ash-2 depletion; n = 20, 25, 31 and 28 worms treated with control, ash-2, seip-1, and ash-2 + seip-1 RNAi, respectively. Data are the mean ± s.d. Each dot represents the lipid droplet number in a 26 × 26 µm² area in the intestine of an individual worm. P values: two-tailed Mann–Whitney test. Lipid droplets shown in Fig. 3f. f,g, Intestinal lipid droplet number, assessed by fluorescence, in dhs-3p::dhs-3::GFP worms following atgl-1 depletion. f, Zoomed-in images of the intestine. Scale bar, 5 µm. g, Quantification of lipid droplet number; n = 20 and 26 worms treated with control and atgl-1 RNAi, respectively. Analysis as in e. h, Atgl-1 knockdown extends lifespan; n ≥ 108 worms for each condition. The percentage of median lifespan extension and P values are indicated. P values: log-rank Mantel–Cox test. a,e,h, Representative of two independent experiments. g, Representative of three independent experiments. Source numerical data of all experiments, replicates and statistics are provided. Source data

Extended Data Fig. 3. BioSorter gating strategy and sorting example.

a, BioSorter sorting example on a synchronized population of adult dhs-3p::dhs-3::GFP worms according to their GFP fluorescence intensity (as a proxy for lipid droplet number). Optical density (extinction) and axial length (time of flight) of all particles measured by the sorter (left). Gate R3 was used to exclude bacterial debris and eggs and enrich for adult worms (15% of all particles). Individual worms from Gate R3 were sorted by GFP fluorescence intensity to separate the highest 10% fluorescent worms (Gate R4; right) and the lowest 10% fluorescent worms (Gate R1; right). b, Intestinal lipid droplet number, assessed in a synchronized population of middle-aged (adult day 6) dhs-3p::dhs-3::GFP worms, after manual sorting. Zoomed-in images of the intestine. Scale bar, 5 µm. Lipid droplet number is quantified in Fig. 4e.

Extended Data Fig. 4. Effect of salinazid treatment on lifespan and number of lipid droplets.

a, Triglyceride abundance among all lipids in middle-aged worms following ash-2 depletion; n = 6 independent biological replicates examined in one experiment. Box-and-whisker plot, with median (central line), 25th and 75th percentile (outer lines) and minimum and maximum within 1.5× the interquartile range (whiskers). Each dot represents one biological replicate. P values: two-tailed Wilcoxon test with Benjamini–Hochberg test for multiple hypothesis correction. b, Salinazid and fat-2 depletion act in the same pathway to extend lifespan; n ≥ 111 worms for each condition. Percentages of median lifespan extension and P values are indicated. P values: log-rank Mantel–Cox test. c, Intestinal lipid droplets, measured by fluorescence, in dhs-3p::dhs-3::GFP worms following Salinazid treatment; n = 29 and 26 worms treated with and without salinazid, respectively. Data are the mean ± s.d. Each dot represents the lipid droplet number in a 26 × 26 µm² area in the intestine of an individual worm. P values: two-tailed Mann–Whitney test. d, Lpin-1 is necessary for longevity following salinazid treatment; n ≥ 105 worms for each condition. Analysis as in b. b–d, Representative of two independent experiments. Source numerical data of all experiments, replicates and statistics as well as Cox proportional hazard interaction values are provided. Source data

Extended Data Fig. 5. Interventions that regulate the number of peroxisomes and lipid droplets.

a, Quantification of intestinal peroxisome-localized GFP intensity, measured by fluorescence, in ges-1p::GFP–SKL worms following MUFA accumulation; n = 6,061, 13,446 and 10,598 peroxisomes in ≥35 worms treated with control, ash-2 and fat-2 RNAi, respectively. Data are the mean ± s.d. Each dot represents the GFP intensity of one peroxisome. P values: two-tailed Mann–Whitney test. Peroxisomes shown in Fig. 7c. b–d, Intestinal peroxisomes, assessed by fluorescence, in worms expressing an integrated array of ges-1p::GFP–SKL or a single copy knock-in of a peroxisome-localized GFP (GFP–SKL) driven by the ubiquitous eft-3 promoter (eft-3p::GFP–SKL) following ash-2 depletion. b, Zoomed-in images of the intestine. Scale bar, 5 µm. c, Quantification of peroxisome number; n = 30, 29, 32 and 27 ges-1p::GFP–SKL worms treated with control and ash-2 RNAi, and eft-3p::GFP-SKL worms treated with control and ash-2 RNAi, respectively. Data are the mean ± s.d. Each dot represents the peroxisome number in a 13 × 13 µm² area in the intestine of an individual worm. P values: two-tailed Mann–Whitney test. d, Quantification of peroxisome-localized GFP intensity; n = 1,922, 2,814, 1,537 and 1,553 peroxisomes in ≥22 ges-1p::GFP–SKL worms treated with control and ash-2 RNAi, and eft-3p::GFP–SKL worms treated with control and ash-2 RNAi, respectively. Analysis in a. e, Quantification of intestinal peroxisomes, measured by fluorescence, in ges-1p::GFP–SKL worms following prx-5 depletion and MUFA accumulation; n = 39, 38, 40, 33, 26 and 22 worms treated with control, ash-2, fat-2, prx-5, ash-2 + prx-5, and fat-2 + prx-5 RNAi, respectively. Data are the mean ± s.d. Each dot represents the peroxisome number in a 26 × 26 µm² area in the intestine of an individual worm. P values: two-tailed Mann–Whitney test. f, prx-5 is necessary for longevity following oleic acid supplementation; n ≥ 120 worms for each condition. Percentages of median lifespan extension and P values are indicated. P values: log-rank Mantel–Cox test. g, Lipid oxidation quantified via MDA levels following prx-5 depletion; n = 8 and 7 samples from worms treated with control and prx-5 RNAi, respectively. Normalized to the control condition. Data are the mean ± s.d. of two independent experiments. Each dot represents a biological replicate. Each shape represents an independent experiment. P values: two-tailed Mann–Whitney test. h, Intestinal lipid droplet number, assessed by fluorescence, in dhs-3p::dhs-3::GFP worms following prx-5 depletion. Zoomed-in images of the intestine. Scale bar, 5 µm. Lipid droplet number quantified in n. i, prx-19 is necessary for longevity following ash-2 depletion; n ≥ 95 worms for each condition. Analysis as in f. j,k, Intestinal lipid droplet and peroxisome number, measured by fluorescence, in dhs-3p::dhs-3::GFP; vha-6p::mRFP–SKL worms during aging and following ash-2 depletion; n ≥ 23 worms. Data are the mean ± s.d. Each dot represents the mean organelle number of all worms imaged for this condition normalized to young control (adult day 1) worms. Organelle number increases at younger ages (j) and organelle number decreases at older ages (k). Linear regression fit. l, Intestinal peroxisomes, measured by fluorescence, in dhs-3p::dhs-3::GFP; vha-6p::mRFP–SKL worms after sorting based on lipid droplet number using a BioSorter; n = 27 and 28 worms sorted based on high and low lipid droplet number, respectively. Analysis as in e. m, Lipid droplets assessed by immunogold labeling (against GFP) using transmission electron microscopy in dhs-3p::dhs-3::GFP worms. The asterisk indicates close proximity/contact between a lipid droplet and the endoplasmic reticulum (top). Scale bar, 500 nm. Quantification of lipid droplet contact/close proximity with other organelles as a percentage (bottom). n, Intestinal lipid droplets, measured by fluorescence, in dhs-3p::dhs-3::GFP; vha-6p::mRFP–SKL transgenic worms following treatment with 62 different RNAis; n = 19–423 worms. Data are the mean ± s.d. Each dot represents the organelle number in a 26 × 26 µm² area in the intestine of an individual worm normalized to control worms. Orange, significant increase in organelle number. Turquoise, significant decrease in organelle number. P values: two-tailed Wilcoxon test with Benjamini–Hochberg test for multiple hypothesis correction. Conditions are colored if the adjusted P < 0.05. *dhs-3 RNAi abolishes the GFP signal of the lipid droplet DHS-3::GFP reporter. o, Quantification of intestinal peroxisome measured by fluorescence in dhs-3p::dhs-3::GFP; vha-6p::mRFP–SKL transgenic worms. Quantification of peroxisome number in worms treated as in n; n = 18–286 worms for each condition. Analysis as in n. p, vps13d depletion does not reduce longevity following oleic acid supplementation; n ≥ 99 for each condition. Analysis as in f. q, rab-7 depletion reduces longevity following oleic acid supplementation; n ≥ 120 for each condition. Analysis as in f. a,e, Representative of three independent experiments. c,d,f,i–m,p,q, Representative of two independent experiments. Source numerical data of all experiments, replicates, exact n values and statistics as well as Cox proportional hazard interaction values are provided. Source data

Extended Data Fig. 6. Schematic of the proposed model for how MUFAs extend lifespan.

MUFA accumulation (for example, following dietary oleic acid supplementation) increases lipid droplet and peroxisome numbers, and both organelles are required for MUFA-induced lifespan extension. This concomitant increase in lipid droplets and peroxisomes requires shared upstream transcription factors (MUFAs and other lipids could also directly serve as substrates for lipid droplets synthesis). Genes encoding proteins implicated in lipid transport (VPS13D/vps13d) and organelle regulation (RAB7/rab-7) are probably involved between these two organelles (because their deficiency uncouples co-regulation of lipid droplet and peroxisome number). MUFAs increase the MUFA-to-PUFA ratio in membrane lipids and decrease ether lipids—a signature predicted to lower lipid oxidation. MUFAs reduce lipid oxidation and preserve cell and membrane integrity in the organism, and this is one way in which MUFAs extend lifespan. Genes encoding proteins that regulate lipid droplet number can also influence lipid oxidation. For example, LIPIN1/lpin-1 deficiency increases lipid oxidation (but not SEIPIN/seip-1). Both LIPIN1/lpin-1 and SEIPIN/seip-1 deficiency reduce MUFA-induced longevity, suggesting that SEIPIN has other beneficial effects, independently of lipid oxidation, perhaps on the endoplasmic reticulum. Salinazid, an iron-chelator, prevents lipid oxidation and acts in a similar pathway as MUFAs (as these are not additive on lifespan extension). Salinazid also increases lipid droplet number, perhaps indirectly or as part of a feedforward mechanism. Peroxisome function drives lipid oxidation (probably by generating reactive oxygen species), suggesting that their beneficial effect for MUFA-induced longevity is also due to other processes (perhaps production of membrane lipids important for membrane integrity or lipids important for metabolism). Although the increase in lipid droplet number seems to be more important than that of peroxisomes, the concomitant upregulation of lipid droplet and peroxisome number in the organism is optimal for lifespan extension. Black arrows, direct effects. Gray arrows, indirect effects. Created with BioRender.com.