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• W4386078275 abstract "Full text Figures and data Side by side Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Abstract Biguanides, including the world’s most prescribed drug for type 2 diabetes, metformin, not only lower blood sugar, but also promote longevity in preclinical models. Epidemiologic studies in humans parallel these findings, indicating favorable effects of metformin on longevity and on reducing the incidence and morbidity associated with aging-related diseases. Despite this promise, the full spectrum of molecular effectors responsible for these health benefits remains elusive. Through unbiased screening in Caenorhabditis elegans, we uncovered a role for genes necessary for ether lipid biosynthesis in the favorable effects of biguanides. We demonstrate that biguanides prompt lifespan extension by stimulating ether lipid biogenesis. Loss of the ether lipid biosynthetic machinery also mitigates lifespan extension attributable to dietary restriction, target of rapamycin (TOR) inhibition, and mitochondrial electron transport chain inhibition. A possible mechanistic explanation for this finding is that ether lipids are required for activation of longevity-promoting, metabolic stress defenses downstream of the conserved transcription factor skn-1/Nrf. In alignment with these findings, overexpression of a single, key, ether lipid biosynthetic enzyme, fard-1/FAR1, is sufficient to promote lifespan extension. These findings illuminate the ether lipid biosynthetic machinery as a novel therapeutic target to promote healthy aging. Editor's evaluation This paper explores the molecular basis underlying metformin treatment to understand why it is such an effective drug for improving age-related health and lifespan. Using C. elegans as a model organism in which to do this, the paper hones in on the role of ether lipid biosynthesis as an effector of metformin, and more broadly as a process implicated in extending lifespan in response to diet, TOR signalling and mitochondrial based interventions. The compelling data substantially support the conclusions and the better understanding of biguanide impact on metabolism is highly important in the field. https://doi.org/10.7554/eLife.82210.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Metformin is the drug most prescribed to treat type 2 diabetes around the world and has been in clinical use since 1950. The drug belongs to a family of compounds known as biguanides which reduce blood sugar, making them an effective treatment against type 2 diabetes. More recently, biguanides have been found to have other health benefits, including limiting the growth of various cancer cells and improving the lifespan and long-term health of several model organisms. Epidemiologic studies also suggest that metformin may increase the lifespan of humans and reduce the incidence of age-related illnesses such as cardiovascular disease, cancer and dementia. Given the safety and effectiveness of metformin, understanding how it exerts these desirable effects may allow scientists to discover new mechanisms to promote healthy aging. The roundworm Caenorhabditis elegans is an ideal organism for studying the lifespan-extending effects of metformin. It has an average lifespan of two weeks, a genome that is relatively easy to manipulate, and a transparent body that enables scientists to observe cellular and molecular events in living worms. To discover the genes that enable metformin’s lifespan-extending properties, Cedillo, Ahsan et al. systematically switched off the expression of about 1,000 genes involved in C. elegans metabolism. They then screened for genes which impaired the action of biguanides when inactivated. This ultimately led to the identification of a set of genes involved in promoting a longer lifespan. Cedillo, Ahsan et al. then evaluated how these genes impacted other well-described pathways involved in longevity and stress responses. The analysis indicated that a biguanide drug called phenformin (which is similar to metformin) increases the synthesis of ether lipids, a class of fats that are critical components of cellular membranes. Indeed, genetically mutating the three major enzymes required for ether lipid production stopped the biguanide from extending the worms’ lifespans. Critically, inactivating these genes also prevented lifespan extension through other known strategies, such as dietary restriction and inhibiting the cellular organelle responsible for producing energy. Cedillo, Ahsan et al. also showed that increasing ether lipid production alters the activity of a well-known longevity and stress response factor called SKN-1, and this change alone is enough to extend the lifespan of worms. These findings suggest that promoting the production of ether lipids could lead to healthier aging. However, further studies, including clinical trials, will be required to determine whether this is a viable approach to promote longevity and health in humans. Introduction Metformin is the first line therapy for type 2 diabetes and the most frequently prescribed oral hypoglycemic medication worldwide (Inzucchi et al., 2012). Human epidemiologic studies note an association between metformin use and decreased incidence of cancer (Evans et al., 2005; Yuan et al., 2013). In addition, metformin extends lifespan in invertebrate and vertebrate models (Cabreiro et al., 2013; Martin-Montalvo et al., 2013; Onken and Driscoll, 2010), and therefore may reduce aging-related diseases in humans (Barzilai et al., 2016). Nonetheless, our understanding of the molecular pathways governing the health-promoting effects of metformin is only just beginning to emerge. Our previous work identified a conserved signaling axis connecting mitochondria, the nuclear pore complex, and mTORC1 inhibition that is required for metformin-mediated extension of lifespan in Caenorhabditis elegans and inhibition of growth in worms and human cancer cells (Wu et al., 2016). The energy sensor AMP-activated protein kinase (AMPK) is not necessary for metformin-induced growth inhibition in C. elegans but is required for the drug’s pro-longevity effects (Cabreiro et al., 2013; Onken and Driscoll, 2010; Chen et al., 2017). Consistently, mechanistic studies indicate that the longevity-promoting transcription factor SKN-1/nuclear factor erythroid 2-related factor (Nrf) is required for biguanide-mediated lifespan extension (Cabreiro et al., 2013; Onken and Driscoll, 2010). The relationship of these metformin longevity response elements to each other and their hierarchy in the biological response to biguanides remains unknown. Thus, the mechanisms by which metformin exacts its beneficial effects on health are likely to be branching and complex. The importance of ether lipids, a major structural component of cell membranes, to aging and longevity is not fully established. Ether lipids are involved in the maintenance of general membrane fluidity and in the formation of lipid rafts within microdomains, which are important for promotion of membrane fusion and cellular signaling (Glaser and Gross, 1994; Komljenovic et al., 2009; Marrink and Mark, 2004). Ether lipids have broad roles in the regulation of cell differentiation (Davies et al., 2001; Facciotti et al., 2012; Rodemer et al., 2003; Teigler et al., 2009), cellular signaling (Thukkani et al., 2002; Albert et al., 2003), and reduction of oxidative stress through their action as antioxidants (Morand et al., 1988; Zoeller et al., 1988; Reiss et al., 1997; Maeba et al., 2002). Humans deficient in ether lipid biogenesis suffer from rhizomelic chondrodysplasia punctata (RCDP), a rare genetic disorder, which results in skeletal and facial abnormalities, psychomotor retardation, and is uniformly fatal typically before patients reach their teenage years (White et al., 2003). Thus, current evidence linking alterations in ether lipid levels to aging and longevity in humans is strictly correlative (Gonzalez-Covarrubias et al., 2013; Pradas et al., 2019). Ether lipids, which are structurally distinct from canonical phospholipids, have a unique biosynthetic pathway through which a fatty alcohol is conjugated to the glycerol backbone at the sn-1 position via an ether linkage. Ether lipid precursors are first synthesized by enzymes associated with the membranes of peroxisomes (Ghosh and Hajra, 1986; Hardeman and van den Bosch, 1989; Singh et al., 1993.) The main enzymes involved in ether lipid biosynthesis within the peroxisomal matrix are glyceronephosphate O-acyltransferase (GNPAT) and alkylglycerone phosphate synthase (AGPS). Fatty acyl-CoA reductase 1 (FAR1) supplies most of the fatty alcohols used to generate the ether linkage in the precursor, 1-O-alkyl-glycerol-3-phosphate. This precursor is then trafficked to the endoplasmic reticulum (ER) for acyl chain remodeling to produce various ether lipid products (Hua et al., 2017). In C. elegans, loss-of-function mutations of any of the three main enzymes involved in human ether lipid biosynthesis, acl-7/GNPAT, ads-1/AGPS, and fard-1/FAR1, result in an inability to produce ether-linked lipids, as in humans, and has been reported to shorten worm lifespan (Drechsler et al., 2016; Shi et al., 2016). Worms and human cells deficient in ether lipids exhibit compensatory changes in phospholipid species, including increases in phosphatidylethanolamines and phosphatidylcholines containing saturated fatty acids (Rodemer et al., 2003; Benjamin et al., 2013). However, in contrast to humans, ether lipid deficient nematodes develop to adulthood at a normal rate, providing an opportunity to determine the biological roles of ether lipids in aging and longevity without pleiotropies associated with developmental rate. Here, we show that the ether lipid biosynthetic machinery is necessary for lifespan extension stimulated by metformin or the related biguanide phenformin in C. elegans. Metabolomic analysis indicates that phenformin treatment drives increases in multiple phosphatidylethanolamine-containing ether lipids through direct biguanide action on C. elegans rather than on the bacterial food source. Interestingly, requirement for the ether lipid biosynthetic genes extends to multiple genetic longevity paradigms, including defective mitochondrial electron transport function (isp-1), defective pharyngeal pumping/caloric restriction (eat-2), and compromises in mTOR complex 1 activation (raga-1). We show that overexpressing fard-1, the enzyme that produces fatty alcohols for ether lipid biogenesis in C. elegans, extends lifespan, supportive of the idea that alterations in the ether lipid landscape alone is sufficient to promote healthy aging. Mechanistically, ether lipids promote longevity downstream of biguanide action through activation of metabolic stress defenses and somatic lipid redistribution driven by the transcription factor SKN-1/Nrf. These data suggest that a heretofore unappreciated role for ether lipids is to enable organismal-level, longevity-promoting stress defenses. Results Genes responsible for ether lipid biosynthesis are necessary for biguanide-induced lifespan extension A prior screen of ~1000 metabolic genes for RNA interference (RNAi) knockdowns that interfere with the growth-inhibitory properties of a high, 160 mM dose of metformin in C. elegans (utilized to maximize the sensitivity and specificity of our assay to identify true epistatic candidates) (Wu et al., 2016), yielded fard-1 and acl-7, which are required for ether lipid biosynthesis. Ether lipids are distinguished from canonical phospholipids as the latter contain exclusively fatty acids conjugated to glycerol, whereas ether lipids contain a fatty alcohol conjugated to the glycerol backbone at the sn-1 position via an ether linkage (Figure 1A). Confirming our screen results, granular, quantitative analysis following RNAi knockdown of fard-1 and acl-7 reveals significant resistance to biguanide-induced growth inhibition (Figure 1—figure supplement 1A). Our lab has previously demonstrated that biguanide effects on growth in C. elegans share significant overlap mechanistically with the machinery by which metformin extends lifespan in the worm, thus suggesting that modulation of ether lipid biosynthesis may also be responsible for the lifespan-extending properties of the drug (Wu et al., 2016). Indeed, loss-of-function mutations in any of three genes encoding enzymes required for ether lipid biosynthesis, fard-1, acl-7, or ads-1, significantly abrogate lifespan extension induced by lifespan-extending doses of metformin (50 mM) and the related biguanide phenformin (4.5 mM) (Figure 1B–G). Loss-of-function of ads-1 and acl-7 may display a modest increase in lifespan with metformin administration but display a percentage median lifespan increase significantly reduced in comparison to wild-type controls (Figure 1B–G, and throughout manuscript see Supplementary file 1 for all tabular survival statistics and biological replicates). Confirming that these mutations confer resistance to metformin by compromising ether lipid synthetic capacity, RNAi knockdowns of fard-1 and acl-7 in wild-type worms also partially impair lifespan extension promoted by phenformin (Figure 1—figure supplement 1B–C). This dependency is not confounded by chemical inhibition of reproduction, as lifespan analyses performed without the use of the thymidylate synthase inhibitor 5-fluoro-2′-deoxyuridine (FUdR) reveal similar abrogation of biguanide-mediated lifespan extension with inactivation of the ether lipid synthetic machinery (Figure 1—figure supplement 2A–F; Van Raamsdonk and Hekimi, 2011). Studies from this point forward are presented predominantly with phenformin because phenformin is more readily absorbed without need for a specific transporter, unlike metformin (Wu et al., 2016; Sogame et al., 2009; Segal et al., 2011), and our experience indicates more consistent lifespan extension with phenformin in C. elegans. Figure 1 with 2 supplements see all Download asset Open asset 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 al., 2016 and Dean and Lodhi, 2018). The latter two are localized to the peroxisomal lumen. (B–D) Missense, loss-of-function mutations in fard-1 (B), acl-7 (C), and ads-1 (D) in C. elegans suppress phenformin-induced lifespan extension. (E–G) A deficiency of ether lipid synthesis in fard-1 (E), acl-7 (F), and ads-1 (G) worm mutants blunts metformin-induced lifespan extension. Results are representative of three biological replicates. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 by log-rank analysis. Note that (B–D) and (E–G) contain the same wild-type (wt) controls as they are visualized from the same replicate of the study. See also Figure 1—figure supplement 1 and refer to Supplementary file 1 for tabular survival data and biological replicates. (H–I) Normalized concentrations of phenformin (H) and metformin (I) in vehicle, 4.5 mM phenformin, or 50 mM metformin-treated wt C. elegans versus fard-1, acl-7, and ads-1 mutants. n=3 biological replicates; ***, p<0.004 by two-tailed Student’s t-test with Bonferroni correction for multiple hypothesis testing. Box represents 75th/25th percentiles, while whisker represents higher/lower hinge ± [1.5 * interquartile range (IQR)]. Because ether lipids are a major structural component of cell membranes, one possibility is that deficiencies in ether lipid synthesis compromises drug action by reducing biguanide bioavailability in the worm. To test this, we compared the relative levels of biguanides present in vehicle- and biguanide-treated wild-type animals to the three ether lipid synthesis mutants by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A comparison of normalized concentrations of phenformin across all four strains shows that phenformin abundance is quantitatively similar across wild-type and the three ether lipid mutant strains (Figure 1H and Figure 1—figure supplement 1D). Similar results were obtained when comparing levels of metformin in wild-type vs. ether lipid mutant animals (Figure 1I and Figure 1—figure supplement 1E). Thus, a deficiency in ether lipid synthesis does not significantly impact levels of biguanides in metformin- and phenformin-treated C. elegans. Phenformin induces changes in ether lipid levels We reasoned that if biguanides require ether lipid biosynthesis to promote lifespan extension, that phenformin may promote synthesis of one or more ether lipids. To investigate the impact of biguanides on ether lipids at a high level, we first utilized gas chromatography-mass spectrometry (GC-MS) analysis. We first recapitulated the observation that fard-1 mutants show absence of 18-carbon containing fatty alcohol derivatives (dimethylacetals [DMAs], which indicate alkenyl ether lipid or plasmalogen levels) and an accumulation of stearate (18:0) relative to wild-type controls by GC-MS (Figure 2A—B; Shi et al., 2016). We then asked if phenformin impacts the levels of 18-carbon alkenyl ether lipids in wild-type animals and if those corresponding changes are absent in fard-1 mutants. Strikingly, phenformin-treated wild-type worms display a significant increase in 18:0 DMA versus vehicle, whereas no such increase is evident in drug-treated fard-1 worms (Figure 2C). In addition, relative proportions of stearic acid (18:0) levels within the total fatty acid pool are significantly increased in fard-1 mutants treated with phenformin versus vehicle-treated fard-1 controls (Figure 2D). In comparison, the relative proportion of stearic acid does not rise in phenformin-treated wild-type animals, suggesting that stearate is being utilized for ether lipid production. Analysis of the total fatty acid pool by GC-MS (Figure 2—figure supplement 1) indicates that aside from several fatty acids (e.g. 18:2), the most pronounced differences were in the plasmalogen pool. In alignment, an assessment of levels of additional alkenyl fatty alcohols in phenformin-treated, wild-type animals indicates a parallel, significant increase in the less abundant 16:0 DMA and 18:1 DMA species (Figure 2E). We conclude that phenformin treatment leads to an overall increase of alkenyl ether lipid levels in C. elegans. Figure 2 with 2 supplements see all Download asset Open asset 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 spectrometry (GC/MS) (A) and accumulation of the saturated fatty acid stearate (18:0, B). (C) Wild-type (wt) worms treated with 4.5 mM phenformin display a significant increase in 18:0 DMA relative to vehicle control, indicative of higher levels of alkenyl ether lipids, with levels remaining essentially undetectable in fard-1 mutants on vehicle or drug. (D) Phenformin (4.5 mM) treatment does not impact stearate levels in wt worms, however it does result in a greater accumulation of stearate in fard-1 mutants. For (A–D), **, p<0.01; ****, p<0.0001, by t-test (A–B) or two-way ANOVA (C–D), n=3 biological replicates. (E) Phenformin (4.5 mM) treatment results in a significant increase in 16:0 DMA and 18:1 DMA in wt worms, relative to vehicle-treated controls *, p<0.05; **, p<0.01, by multiple t-tests, with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. n=3 biological replicates. (F) Heatmap of normalized ether lipid abundance following phenformin treatment in wt C. elegans indicates an overall increase in ether lipids relative to vehicle-treated controls, and this shift is absent in ether lipid deficient mutants. All metabolites shown have an FDR adjusted p<0.05 by one-way ANOVA followed by Fisher’s LSD post hoc testing for wt versus fard-1, ads-1, and acl-7 mutants. (G) Liquid chromatography-tandem mass spectrometry (LC-MS) analysis shows that phosphatidylethanolamine-containing ether lipids detected exhibited a general trend toward increased abundance in wild-type worms treated with 4.5 mM phenformin. Four of these ether lipids reached statistical significance: PE(O-16:0/18:1), PE(O-18:0/18:3), PE(O-18:0/20:2), and PE(P-18:1/18:1). Eleven of the ether lipids detected are of the alkyl-type (indicated by ‘O’ in their name prior to fatty alcohol designation) whereas nine are of the alkenyl-type (plasmalogen, indicated by ‘P’ in their name prior to the fatty alcohol designation) ether lipids. For (G), *, p<0.05; **, p<0.01; ****, p<0.0001, by multiple t-tests, with multiple hypothesis testing correction by two-stage step-up method of Benjamini, Krieger, and Yekutieli, n=3 biological replicates. See Figure 2—source data 1 for raw and normalized mass spectrometry data. 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 Download elife-82210-fig2-data1-v1.xlsx To investigate relative changes in individual ether lipid abundance in response to phenformin at high resolution, we utilized LC-MS/MS analysis. Using this method, we detected 20 alkyl and alkenyl phosphatidylethanolamine-based ether lipids previously noted to be the most abundant ether lipids in C. elegans (Drechsler et al., 2016; Shi et al., 2016; Figure 2F–G and Figure 2—source data 1). This analysis indicates that phenformin treatment results in a significant increase in normalized abundance of four ether lipids, PE(O-16:0/18:1), PE(O-18:0/18:3), PE(O-18:0/20:2), and PE(P-18:1/18:1), even when corrected for multiple hypothesis testing. Most ether lipids measured display mean levels that increase with phenformin treatment, though these changes are either nominally significant or exhibit a nonsignificant trend because of the strict threshold required to reach significance when correcting for multiple hypotheses. Finally, phosphatidylethanolamine ether lipid abundances were extremely low in fard-1, acl-7, and ads-1 mutants and unchanged by phenformin treatment, unlike in wild-type animals (Figure 2F and Figure 2—source data 1). In aggregate, these data indicate that phenformin treatment leads to increased abundance of multiple ether lipid species in C. elegans. Peroxisomal ether lipid synthesis is essential to the biological action of phenformin In order to begin to understand the governance of ether lipid biosynthesis by biguanides, we examined the expression of a C. elegans FARD-1::RFP translational reporter, under the control of its own promoter (Figure 2—figure supplement 2A). Exogenously expressed FARD-1 (fard-1 oe1) is expressed in the intestine and localizes near structures resembling lipid droplets by Nomarski microscopy (Figure 2—figure supplement 2B). Given that ether lipid biogenesis occurs between peroxisomes and the ER (Ghosh and Hajra, 1986; Hardeman and van den Bosch, 1989; Singh et al., 1993; Hua et al., 2017), we crossed this FARD-1::RFP reporter to an animal bearing a GFP reporter that illuminates peroxisomes in the intestine (GFP fused to a C-terminal peroxisomal targeting sequence 1 [PTS1]) to determine if localization of FARD-1 is regulated by biguanides. FARD-1 does not possess a predicted PTS, in contrast to ACL-7 and ADS-1. At baseline, FARD-1::RFP fluorescence partially overlaps with peroxisomally targeted GFP (Figure 2—figure supplement 2C). Colocalization analysis indicates that treatment with phenformin does not change the amount of overlap between FARD-1::RFP and GFP::PTS1 relative to vehicle-treated controls (Figure 2—figure supplement 2D). To confirm our earlier observation that suggests FARD-1 colocalization with lipid droplets, we used confocal imaging to assess the spatial distribution of an integrated FARD-1::RFP reporter (fard-1 oe3) in C. elegans fed C1-BODIPY-C12 to label lipid droplets (and treated with glo-4 RNAi to remove BODIPY-positive lysosome-related organelles) (Hermann et al., 2005; Zhang et al., 2010b; Zhang et al., 2010a). We found that FARD-1::RFP fluorescence directly surrounds some, but not all, BODIPY-positive lipid droplets in the worm intestine (Figure 2—figure supplement 2E). However, as with peroxisomes, phenformin does not alter the number of lipid droplets that are surrounded by FARD-1 or its distribution around lipid droplets (data not shown). Finally, FARD-1::RFP localizes into web-like structures in the fard-1(oe3) reporter that may represent smooth ER versus another cellular tubular vesicular network (Figure 2—figure supplement 2F), and this localization is also not altered by biguanide treatment. Thus, the regulation of ether lipid biosynthesis does not appear to be via differential localization of FARD-1. We next examined expression of mRNAs encoding FARD-1, ACL-7, and ADS-1 following biguanide treatment. Each of these mRNAs decreased or remain unchanged in abundance upon treatment with biguanide via quantitative RT-PCR (Figure 2—figure supplement 2G–L), suggesting that ether lipids are not increased in phenformin treatment through a transcriptional mechanism. A parallel decrease in overall levels of FARD-1::RFP protein of fard-1(oe1) transgenics was seen with phenformin treatment (Figure 2—figure supplement 2M). These seemingly paradoxical data are likely consistent with post-translational negative feedback of ether lipids on the ether lipid biosynthetic pathway, as has been previously reported (Honsho et al., 2010). To affirm that the peroxisome is an essential site of ether lipid production in biguanide action, we disrupted peroxisomal protein targeting and examined phenformin-stimulated lifespan extension. Indeed, either prx-5 or prx-19 RNAi impair lifespan extension prompted by phenformin fully or partially, respectively (Figure 3A–B). PRX-5 is involved in protein import into the peroxisomal matrix and PRX-19 is involved in proper sorting of proteins for peroxisomal biogenesis. Thus, either disruption of ether lipid biosynthetic machinery or of a principal site of ether lipid biosynthesis impairs phenformin’s pro-longevity benefit. Figure 3 Download asset Open asset 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) and polyunsaturated fatty acid (PUFA) synthesis pathway in C. elegans (adapted from Figure 1 of Watts, 2016). (D–G) RNAi of two fatty acid desaturases (D–E) and two fatty acid elongases (F and G) involved in the synthesis of 18- and 20-carbon PUFAs blunt phenformin-mediated lifespan extension in wild-type worms. Colored symbols for elo and fat genes (vs. those in black and white) in (C) indicates those that inhibit phenformin lifespan extension when knocked down by RNAi. For (A, B) and (D–G), results are representative of two to three biological replicates. **, p<0.01; ***, p<0.001; ****, p<0.0001 by log-rank analysis. Note that (D–G) contain the same wild-type controls as they are visualized from the same replicate of the study. See also Supplementary file 1 for tabular survival data and biological replicates. Fatty acid elongases and desaturases are positive effectors of biguanide-mediated lifespan extension Most mature ether lipid species contain a fatty acid in the sn-2 position linked by an ester bond (Dean and Lodhi, 2018). The majority of fatty acids conjugated in ether lipids are largely synthesized endogenously in C. elegans by fatty acid desaturases and fatty acid elongases (Perez and Van Gilst, 2008; Perez and Watts, 2021; Figure 3C). Thus, we hypothesized that some of these desaturases and elongases may also contribute mechanistically to biguanide-mediated lifespan extension. Indeed, RNAi knockdown of two fatty acid desaturases and two fatty acid elongases in phenformin-treated C. elegans blunted phenformin-stimulated lifespan extension relative to empty vector controls (Figure 3D–G). Notably, these four genes all contribute to the production of fatty acids 18–20 carbons in length with three or more double bonds. Although knockdown of fatty acid desaturases and elongases in C. elegans results in inherent lifespan extension on vehicle relative to wild-type controls on empty vector RNAi as has been previously reported (Shmookler Reis et al., 2011; Horikawa et al., 2008), RNAi knockdown of fat-3, fat-4, elo-1, and elo-2 mitigate phenformin-driven lifespan extension (Figure 3D–G). These results suggest the tantalizing possibility that specific fat" @default.
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• W4386078275 title "Decision letter: Ether lipid biosynthesis promotes lifespan extension and enables diverse pro-longevity paradigms in Caenorhabditis elegans" @default.
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