Ether lipid biosynthesis promotes lifespan extension and enables diverse pro-longevity paradigms in Caenorhabditis elegans
- Center for Genomic Medicine and Diabetes Unit, Endocrine Division, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, United States
- Broad Institute of Harvard and MIT, United States
- Program in Biological and Biomedical Sciences, Division of Medical Sciences, Harvard Medical School, United States
- Leonard Davis School of Gerontology, University of Southern California, United States
- Department of Medicine and Pharmacology, University of California San Diego, United States
- Biomedical Informatics Core, Massachusetts General Hospital and Harvard Medical Schoo, United States
- Program in Innate Immunity, Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, United States
Figures
Figure 1 with 2 supplements
Genes responsible for ether lipid biosynthesis are necessary for biguanide-induced lifespan extension.
(A) C. elegans ether lipid synthesis is catalyzed by three enzymes: fatty acyl reductase FARD-1, acyltransferase ACL-7, and alkylglycerone phosphate synthase ADS-1 (adapted from Figure 1 of Shi et …
Figure 1—figure supplement 1
Reduced function of genes responsible for ether lipid biosynthesis partially suppresses biguanide effects of growth and lifespan without affecting biguanide levels.
(A) RNA interference (RNAi) knockdown of fard-1 and acl-7 induces resistance to growth inhibition by 160 mM metformin treatment in C. elegans. *, p<0.05, by two-way ANOVA, n=2 biological replicates. …
Figure 1—figure supplement 2
The use of 5-fluoro-2′-deoxyuridine (FUdR) in lifespan analyses does not impact the observed epistases between the ether lipid machinery and biguanide-mediated lifespan extension.
Lifespans performed without the use of FUdR to inhibit progeny formation corroborate that a deficiency of ether lipid synthesis in fard-1 (A/D), acl-7 (B/E), and ads-1 (C/F) worm mutants negates …
Figure 2 with 2 supplements
Phenformin treatment of C. elegans leads to increased abundance of multiple alkyl and alkenyl ether lipids.
(A–B) Loss-of-function fard-1 mutants have significant reduction in 18:0 fatty alcohols derivatized from 18-carbon containing alkenyl ether lipids (dimethylacetal [DMA]) by gas chromatography/mass …
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Figure 2—source data 1
Excel file containing raw, normalized, and normalized and log10 transformed mass spectrometry data for phosphatidylethanolamine containing ether lipids detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Data from three biological replicates are shown for molecules indicated for vehicle or 4.5 mM phenformin treatment, for four different genetic backgrounds: wild-type animals (N2, wt), BX10 (ads-1 mutant), BX259 (acl-7 mutant), and BX275 (fard-1 mutant). Compound identity for each detected lipid as well as raw, normalized, or transformed mass counts on each of three tabs. Note, several of the lipids were not uniformly detected or of low abundance, and thus were filtered by the MetaboAnalyst parameters used and not represented on the ‘Normalized’ and ‘Normalized-Log10 Transformed’ tabs.
- https://cdn.elifesciences.org/articles/82210/elife-82210-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Biguanide treatment modulates abundance of fatty acids in C. elegans.
A comparison of the percent of the total fatty acid pool for 33 fatty acids shows that 7 fatty acids are significantly altered in phenformin-treated wild-type worms. n=3 biological replicates. *, p<0…
Figure 2—figure supplement 2
FARD-1::RFP localizes to intestinal lipid droplets and peroxisomes and is not positively regulated at the RNA or protein level by phenformin.
(A) Schematic representation of the C. elegans FARD-1::RFP overexpression reporter. (B) FARD-1::RFP (fard-1 oe1) exhibits intestinal expression in C. elegans. FARD-1 displays a cytoplasmic …
Figure 3
Peroxisomal protein import, fatty acid elongases, and fatty acid desaturases are required for the pro-longevity effects of biguanides.
(A–B) Knockdown of prx-5 (A) and prx-19 (B) by RNA interference (RNAi) eliminates or significantly suppresses phenformin-mediated lifespan extension. (C) Schematic representation of the mono- (MUFA) …
Figure 4 with 1 supplement
Genes involved in ether lipid biosynthesis are required for lifespan extension in multiple longevity paradigms.
(A–C) isp-1, raga-1, and eat-2 mutants display extended lifespan relative to wild-type animals that is dependent upon the three members of the ether lipid biosynthetic pathway. Results are …
Figure 4—figure supplement 1
Ether lipid biosynthetic genes are not necessary for daf-2-dependent lifespan extension.
daf-2 mutants display extended lifespan relative to wild-type animals. RNA interference (RNAi) knockdown of fard-1, acl-7, and ads-1 does not impact lifespan extension in these mutants. ns, p>0.05; …
Figure 5 with 1 supplement
fard-1 overexpression is sufficient to extend lifespan by modulating ether lipid synthesis.
(A–B) Two independently generated fard-1 overexpression (fard-1 oe1 and fard-1 oe2) transgenic strains exhibit lifespan extension that is not further extended by concomitant phenformin treatment. (C)…
Figure 5—figure supplement 1
fard-1 overexpression extends lifespan in a manner dependent upon ether lipid biosynthesis, and not apparently involving ferroptosis.
(A–B) RNA interference (RNAi) knockdown of fard-1 (A) and acl-7 (B) suppresses fard-1 overexpression(oe1)-associated lifespan extension. (C–E) Independent knockdown of glutathione peroxidases, gpx-1 …
Figure 6 with 3 supplements
Phenformin modulates systemic lipid metabolism through an ether lipid-skn-1 signaling relay.
(A) The number of intestinal, C1-BODIPY-C12 labeled lipid droplets are significantly lower in day 1 adult phenformin-treated animals versus vehicle (FARD-1::RFP reporter transgenic [fard-1 oe3] …
Figure 6—figure supplement 1
Biguanides do not activate gst-4 expression irrespective of bacterial diet.
(A–B) GFP quantification of gst-4p::NLS::GFP animals treated from hatching with vehicle, 50 mM metformin, or 4.5 mM phenformin on either OP50-1 seeded nematode growth media (NGM) plates or EV HT115 …
Figure 6—figure supplement 2
Disruption of bacterial growth and metabolism does not prevent biguanide-mediated induction of ether lipid synthesis.
(A) Bacterial titer assay measuring viability of OP50-1 treated with standard seeding conditions (live OP50-1), treated with 1% phosphate buffered saline (PBS) for 2 hr (mock-treated OP50-1 [2 hr]), …
Figure 6—figure supplement 3
Inactivation of ether lipid machinery disrupts biguanide-mediated lifespan extension independent of effects on bacterial growth or metabolism.
Lifespan analyses of wild-type (wt) or ads-1 mutant animals grown on live OP50-1 (A–B), mock-treated OP50-1 for 2 hr (C–D), or 1% paraformaldehyde (PFA)-treated OP50-1 for 2 hr (E–F) reveal that ads-…
Figure 7
Schematic representation for the role of the ether lipid biosynthetic machinery in multiple pro-longevity paradigms.
(A) Model of ether lipid action in biguanide-prompted lifespan extension. Activation of ether lipid biosynthesis leads to longevity-promoting activity of metabolic stress defenses downstream of the …
Author response image 1
Gain of function mutation in SKN-1 results in global depletion of free fatty acid levels and elevates ether lipid precursor alcohols.
(A) Quantification of total area under the curve (AUC) measurements for all identified free fatty acids (FFA) in wildtype (N2) and skn-1gf(lax188) animals at Adult Day 2 using fatty acid methyl …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | OP50-1 | Caenorhabditis Genetics Center | RRID: WB-STRAIN:WBStrain00041971 | Standard laboratory stock |
| Strain, strain background (Escherichia coli) | HT115(DE3) | Caenorhabditis Genetics Center | RRID: WB-STRAIN:WBStrain00041079 | Background strain for RNAi clones utilized from Ahringer and Vidal Libraries |
| Strain, strain background (Caenorhabditis elegans) | Bristol N2 (wt) | Caenorhabditis Genetics Center | RRID: WB-STRAIN:WBStrain00000001 | Standard laboratory wild-type strain |
| Strain, strain background (Caenorhabditis elegans) | fard-1(wa28) [G261D] | Caenorhabditis Genetics Center | RRID: WB-STRAIN:WBStrain00004025 | BX275 |
| Strain, strain background (Caenorhabditis elegans) | acl-7(wa20) [R234C] | Caenorhabditis Genetics Center | RRID: WS-STRAIN:WBStrain00004024 | BX259 |
| Strain, strain background (Caenorhabditis elegans) | ads-1(wa3) [G454D] | Caenorhabditis Genetics Center | RRID: WB-STRAIN:WBStrain00004007 | BX10 |
| Strain, strain background (Caenorhabditis elegans) | daf-2(e1370) | Caenorhabditis Genetics Center | RRID: WB-STRAIN:WBStrain00004309 | CB1370 |
| Strain, strain background (Caenorhabditis elegans) | isp-1(qm150) | Caenorhabditis Genetics Center | RRID: WB-STRAIN:WBStrain00026672 | MQ989 |
| Strain, strain background (Caenorhabditis elegans) | raga-1(ok701) | Caenorhabditis Genetics Center | RRID: WB-STRAIN:WBStrain00035849 | VC533 |
| Strain, strain background (Caenorhabditis elegans) | eat-2(da465) | Caenorhabditis Genetics Center | RRID: WB-STRAIN:WBStrain00005463 | DA465 |
| Strain, strain background (Caenorhabditis elegans) | mgIs43[ges-1p::GFP::PTS1] | Soukas Laboratory | N/A | MGH48 |
| Strain, strain background (Caenorhabditis elegans) | skn-1(lax188) | Caenorhabditis Genetics Center | RRID: WB-STRAIN:WBStrain00034420 | skn-1gf, SPC168 |
| Strain, strain background (Caenorhabditis elegans) | agIs6[dod-24p::GFP] | Caenorhabditis Genetics Center | RRID: WB-STRAIN:WBStrain00004921 | CF3556 |
| Strain, strain background (Caenorhabditis elegans) | dvIs19[(pAF15)gst-4p::GFP::NLS] | Caenorhabditis Genetics Center | RRID: WB-STRAIN:WBStrain00005102 | CL2166 |
| Strain, strain background (Caenorhabditis elegans) | skn-1(zu135) | Caenorhabditis Genetics Center | RRID: WB-STRAIN:WBStrain00007251 | skn-1lf, EU31 |
| Genetic reagent (Caenorhabditis elegans) | alxEx122[fard-1p::FARD-1::mRFP::HA unc-54 3'UTR myo-2p::GFP] | This study | MGH471 | fard-1 (oe1) |
| Genetic reagent (Caenorhabditis elegans) | alxEx135[fard-1p::FARD-1::mRFP::HA unc-54 3'UTR myo-2p::GFP] | This study | MGH472 | fard-1 (oe2) |
| Genetic reagent (Caenorhabditis elegans) | alxIs45[fard-1p::FARD-1::mRFP::HA::unc-54 3'UTR myo-2p::GFP] | This study | MGH605 | fard-1 (oe3), backcrossed into N2 8× |
| Genetic reagent (Caenorhabditis elegans) | alxIs46[fard-1p::FARD-1::mRFP::HA::unc-54 3’UTR myo-2p::GFP] | This study | MGH606 | fard-1 (oe4), backcrossed into N2 8× |
| Genetic reagent (Caenorhabditis elegans) | mgIs43[ges-1p::GFP::PTS1]; alxEx122[fard-1p::FARD-1::mRFP::HA::unc-54 3’UTR myo-2p::GFP] | This study | MGH607 | GFP::PTS1; FARD-1::RFP, prepared by crossing MGH48 into MGH471 |
| Sequence-based reagent | 5’-TGCATGCCTGCAGGTCGACTTTGACAAAAGTTCTGTTGCCG-3’ | This study | AS-4524 | Forward primer used to generate fard-1 overexpression construct |
| Sequence-based reagent | 5’-TTTGGGTCCTTTGGCCAATCGCTTTTTTGAAGATACCGAGAATAATCC-3’ | This study | AS-4527 | Reverse primer used to generate fard-1 overexpression construct |
| Sequence-based reagent | 5’-TGCTGATCGTATGCAGAAGG-3’ | This study | act-1 F | qRT-PCR Primer |
| Sequence-based reagent | 5’-TAGATCCTCCGATCCAGACG-3’ | This study | act-1 R | qRT-PCR Primer |
| Sequence-based reagent | 5’-GTTCCCGTGTTCATCACTCAT-3’ | This study | pmp-3 F | qRT-PCR Primer |
| Sequence-based reagent | 5’-ACACCGTCGAGAAGCTGTAGA-3’ | This study | pmp-3 R | qRT-PCR Primer |
| Sequence-based reagent | 5’-ACAAGTCACCAATGGCTCCAC-3’ | This study | fard-1 F | qRT-PCR Primer |
| Sequence-based reagent | 5’-GCTTTGGTCAGAGTGTAGGTG-3’ | This study | fard-1 R | qRT-PCR Primer |
| Sequence-based reagent | 5’-cgatagtgtgtctgttgattgtga-3’ | This study | fard-1 F (Native 3’ UTR) | qRT-PCR Primer |
| Sequence-based reagent | 5’-agttattgttgatgagagagtgcg-3’ | This study | fard-1 R (Native 3’ UTR) | qRT-PCR Primer |
| Sequence-based reagent | 5’-GTTTATGGCTGGCGTGTTG-3’ | This study | acl-7 F | qRT-PCR Primer |
| Sequence-based reagent | 5’-CGGAGAAGACAGCCCAGTAG-3’ | This study | acl-7 R | qRT-PCR Primer |
| Sequence-based reagent | 5’-GCGATTAACAAGGACGGACA-3’ | This study | ads-1 F | qRT-PCR Primer |
| Sequence-based reagent | 5’-CGATGCCCAAGTAGTTCTCG-3’ | This study | ads-1 R | qRT-PCR Primer |
| Chemical compound, drug | C1-BODIPY-C12 (green) | Invitrogen | Cat#D-3823 | N/A |
| Chemical compound, drug | 5-fluoro-2′-deoxyuridine (FUdR) | Fisher Scientific | Cat#F10705 | N/A |
| Chemical compound, drug | Metformin hydrochloride | MilliporeSigma | Cat#PHR1084 | N/A |
| Chemical compound, drug | Phenformin hydrochloride | MilliporeSigma | Cat#PHR1573 | N/A |
| Commercial assay or kit | Quantitect Reverse Transcription Kit | QIAGEN | Cat#205314 | N/A |
| Commercial assay or kit | Quantitect SYBR Green PCR Reagent | QIAGEN | Cat#204145 | N/A |
| Chemical compound, drug | Levamisole | MilliporeSigma | Cat#L9756 | N/A |
| Software, algorithm | OASIS2 | Structural Bioinformatics Laboratory, POSTECH | https://sbi.postech.ac.kr/oasis2/surv/ | N/A |
| Software, algorithm | MetaMorph | Molecular Devices | https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy | N/A |
| Software, algorithm | Xcalibur (v4.1.31.9) | Thermo Fisher Scientific | Cat#OPTON-30965 | N/A |
| Software, algorithm | QualBrowser (v4.1.31.9) | Thermo Fisher Scientific | Cat#XCALI-97617 | N/A |
| Software, algorithm | MZmine (v2.36) | Open Source | RRID: SCR_012040, http://mzmine.github.io | N/A |
| Software, algorithm | MetaboAnalyst (v5.0) | N/A | https://www.metaboanalyst.ca | N/A |
| Software, algorithm | CellProfiler (v4.2.1) | Broad Institute | https://cellprofiler.org | N/A |
| Software, algorithm | Prism (v9.0) | GraphPad by Dotmatics | https://www.graphpad.com/ | N/A |
| Software, algorithm | Fiji/ImageJ2 (v2.13.1) | NIH | https://imagej.net/software/fiji/ | N/A |
Additional files
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Supplementary file 1
Tabular and survival data including three biological replicates (unless otherwise noted) are shown for lifespan experiments related to Figures 1 and 3—5, Figure 1—figure supplement 1, Figure 1—figure supplement 2, Figure 4—figure supplement 1, Figure 5—figure supplement 1, and Figure 6—figure supplement 3.
Data present a summary of the conditions tested which, if applicable, include: (1) drug treatment with vehicle control and 4.5 mM phenformin or 50 mM metformin and/or (2) RNAi treatment to knockdown expression of the specific denoted gene. The C. elegans strain, number of subjects, restricted mean (days), standard error, 95% confidence interval (CI), 95% median CI, and p-values for relevant comparisons are noted among all conditions. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 by log-rank analysis.
- https://cdn.elifesciences.org/articles/82210/elife-82210-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/82210/elife-82210-mdarchecklist1-v1.docx
