Corrigendum: Deguelin promotes longevity and healthspan through C. elegans fmo-4
Tuckowski AM, Huang S, Chambers K, Buscher B, Leiser SF. 2025. Deguelin promotes longevity and healthspan through C. elegans fmo-4. microPublication Biology. 10.17912/micropub.biology.001548
In the originally published version of this article, the survival curves in Figure 1E were plotted with incorrect line styles and colors. The wild-type deguelin condition, which should appear as a dashed black line, was instead drawn as a solid green line, leaving the plotted conditions mismatched to the legend.
In the corrected Figure 1E, the four conditions are plotted as follows: wild type + DMSO, solid black line; wild type + deguelin, dashed black line; fmo-4 knockout + DMSO, solid green line; fmo-4 knockout + deguelin, dashed green line.
The authors have corrected Figure 1E. The error affected only the graphical display of the data and does not alter the underlying results, statistical analyses, or conclusions of the article.
This error has been corrected online as well as in future PDF versions of this article.
","date":"Mon Aug 31 2026","correctionType":"corrigendum"}],"versions":[{"id":"4e17a2a8-dd42-4ef8-8735-cad6c5946376","decisionLetter":null,"decision":"edit","submitted":true,"abstract":"There are multiple approaches to longevity interventions in Caenorhabditis elegans, including genetic factors that are necessary or sufficient for lifespan extension and pharmacological agents that modify physiology to extend lifespan. Many pharmacological interventions act through known genetic pathways to promote longevity. Here, we show that the mitochondrial complex I inhibitor, deguelin, promotes lifespan extension and healthspan in an fmo-4-dependent manner. Our results confirm that deguelin increases lifespan and indicate that deguelin induces and requires multiple FMO enzymes to extend lifespan in C. elegans, suggesting these enzymes may promote longevity in a coordinated fashion.
","acknowledgements":"This work was supported by grants from NIH. AMT was supported by NIH T32AG000114 and the University of Michigan Rackham Research Grant. SFL was supported by R01AG075061 and the Glenn Foundation for Medical Research.
","authors":[{"affiliations":["University of Michigan"],"credit":["conceptualization","formalAnalysis","investigation","methodology","writing_originalDraft","writing_reviewEditing"],"email":"atuckow@umich.edu","firstName":"Angela M","lastName":"Tuckowski","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9125-4780"},{"affiliations":["Kansas State University"],"credit":["conceptualization","writing_reviewEditing"],"email":"shijiaoh@ksu.edu","firstName":"Shijiao","lastName":"Huang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["methodology","writing_reviewEditing"],"email":"chamberskelly5698@gmail.com","firstName":"Kelly","lastName":"Chambers","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["methodology","writing_reviewEditing"],"email":"bubrando@umich.edu","firstName":"Brandon","lastName":"Buscher","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["writing_reviewEditing","supervision","resources"],"email":"leiser@umich.edu","firstName":"Scott F","lastName":"Leiser","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"comments":"","dataTable":null,"disclaimer":true,"funding":"This work was supported by grants from NIH. AMT was supported by NIH T32AG000114, NIH 5T32GM007315, and the University of Michigan Rackham Research Grant. SFL was supported by R01AG075061 and the Glenn Foundation for Medical Research.
","image":{"name":"Figure micropub Tuckowski.jpg","url":"https://portal.micropublication.org/uploads/a7dd596a42c4239875c87f451fbdf812.jpg"},"imageCaption":"(A) Lifespan assessment of wild-type (WT) worms exposed to DMSO control, 1 µM deguelin, 5 µM deguelin, or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis). (B) Healthspan analysis of wild-type (WT) worms exposed to DMSO control or 10 µM deguelin thrashing in a drop of M9 solution for 30 seconds on days 1 and 8 of adulthood (n = ~10 worms per condition, three replicate experiments). (C) Fluorescence intensity of fmo-4p::mCherry transcriptional reporter worms exposed to DMSO control or 10 µM deguelin (n = ~20 worms per condition, three replicate experiments), quantified in (D). (E) Lifespan assessment of wild-type (WT) and fmo-4 knockout (KO) worms exposed to DMSO control or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis and significant interactions between the condition of interest and genotype was determined at p < 0.01 using Cox regression analysis). (F) Healthspan analysis of wild-type (WT) and fmo-4 knockout (KO) worms exposed to DMSO control or 10 µM deguelin thrashing in a drop of M9 solution for 30 seconds on days 1 and 8 of adulthood (n = ~10 worms per condition, three replicate experiments). (G) Lifespan assessment of wild-type (WT) and mcu-1 knockout (KO) worms exposed to DMSO control or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis and significant interactions between condition of interest and genotype was determined at p < 0.01 using Cox regression analysis). For healthspan and imaging experiments, * denotes significant change at p < 0.05 using unpaired two-tailed t test. NS = not significant. All replicate data can be found in the Source Data files.
","imageTitle":"Deguelin requires fmo-4 to promote lifespan extension and improve healthspan in C. elegans.
","laboratory":{"name":"","WBId":""},"methods":"Strains and Maintenance
Standard C. elegans cultivation procedures were used as previously described3. Worm strains were maintained on solid nematode growth medium (NGM) using E. coli OP50 throughout life. Worms were transferred using a platinum wire. All worm strains were maintained at 20°C.
Lifespan Assays
Gravid adult worms were placed on NGM plates seeded with E. coli OP50 for three hours. Then the adults were removed and eggs were allowed to hatch and develop to day 1 adulthood at 20°C. Adult worms were transferred to NGM plates containing 25 µg/mL carbenicillin, floxuridine (FUdR), and either dimethylsulfoxide (DMSO), 1 µM deguelin (Sigma, D0817), 5 µM deguelin, or 10 µM deguelin. Additionally, these plates were seeded with 200 µL of paraformaldehyde (PFA) killed E. coli OP50 at a concentration of 3x. Approximately 70 worms were transferred to fresh plates on day 1, day 2, day 4, and day 6 of adulthood. Two plates per strain per condition were tested per replicate experiment. Experimental animals were scored every 2-3 days and considered dead when they did not move in response to prodding under a dissection microscope. Worms that crawled off the plate were not considered, but ruptured worms were considered as previously described3. Three replicates were performed for each lifespan assay.
Thrashing Assay
Worms were synchronized by placing 10 gravid adult worms on NGM plates seeded with E. coli OP50 and allowing them to lay eggs for 2 hours at 20°C. The gravid adults were removed and the eggs were allowed to hatch and develop at 20°C until larval stage 2 (L2). At this stage, the L2 worms were transferred to NGM plates containing either DMSO or 10 µM deguelin, seeded with paraformaldehyde killed E. coli OP50. On day 1 adulthood, worms were placed in a drop of M9 solution, as previously described. The body bends were counted at maximum rate for 30 seconds. Thrashing was assayed on day 1 and day 8 of adulthood. The worms that were not used for the day 1 assay were transferred to fresh plates containing either DMSO or deguelin two times until they were ready to be assayed. Three replicates were performed. Data were analyzed in GraphPad Prism using unpaired two-tailed t tests with Welch’s correction.
fmo-4 Induction on Deguelin
Gravid fmo-4p::mCherry transcriptional reporter adult animals were placed on NGM plates seeded with E. coli OP50. After 3 hours, adults were removed and the eggs were allowed to develop at 20°C until they reached larval stage 4 (L4). Then 30 of the L4 worms were transferred to NGM plates containing either DMSO or 10 µM deguelin and seeded with paraformaldehyde (PFA) killed E. coli OP50. The worms were incubated for 24 hours at 20°C. Then ~20 worms per condition were picked off these plates and added to unseeded NGM plates, anesthetized in 0.5 M sodium azide (Sigma), and imaged at 6.3x magnification with the LASx software and Leica scope using the mCherry fluorescence channel. Three replicates were performed. Each worm was measured for fluorescence in ImageJ. Data were analyzed in GraphPad Prism using t tests.
Statistical Analyses
Log-rank test was used to derive p-value for lifespan assays using p < 0.05 cut-off threshold compared to DMSO or wild-type controls. Cox regression was also used to assess interactions between genotype and condition for lifespans using p < 0.01 cut-off threshold compared to controls. Supplemental Data 1 provide the results of the Log-rank test and Cox regression analyses, which were run in RStudio.
Strain Name | Genotype | Source |
WT or N2 | WT | CGC |
RB562 (fmo-4 knockout) | fmo-4(ok294) | CGC |
CZ19982 (mcu-1 knockout) | mcu-1(ju1154) | CGC |
fmo-4 transcriptional reporter | fmo-4p::mCherry | Suny Bioscience |
Reagent | Source | Catalog # |
Deguelin | Sigma | D0817-5MG |
DMSO | Fisher Scientific | BP213-100 |
Sodium Azide | Sigma | S2002-5G |
Aging is a complex biological process that affects all living organisms, leading to a decline in physiological functions and an increased susceptibility to diseases1. Studying the aging process is crucial because it allows for a better understanding of how to prevent or delay the onset of multiple age-related chronic diseases simultaneously. The nematode Caenorhabditis elegans (C. elegans) has emerged as a powerful model organism for studying aging due to its short lifespan, well-characterized genetics, and conserved aging pathways2. Recent research has focused on identifying longevity interventions in C. elegans, including genetic factors and pharmacological agents that can extend lifespan and improve healthspan, potentially offering insights into interventions that could promote healthy aging in humans.
A gene family of particular interest are the flavin-containing monooxygenase (fmo) genes. From this family, fmo-2 and fmo-4 are both sufficient and necessary for lifespan extension in C. elegans3,4. These genes play crucial roles in altering cellular metabolism, including one carbon metabolism5 and calcium regulation between the endoplasmic reticulum and mitochondria4, contributing to the longevity phenotype. Pharmacologically, compounds like deguelin, a natural rotenoid isolated from plants of the Leguminosae family, have gained attention for their potential to extend lifespan through the manipulation or inhibition of cellular processes6-8. For instance, deguelin treatment has been successfully tested as an anti-tumor agent in human cells lines, inhibits mitochondrial complex I, inhibits mammalian target of rapamycin complex I (mTORC1), and extends lifespan in wild-type (WT) C. elegans6-8.
Our lab previously demonstrated that these interventions can act in the same genetic pathway to extend lifespan. Specifically, we have shown that deguelin requires fmo-2 to promote longevity in C. elegans, suggesting that fmo-2 acts downstream of deguelin-mediated lifespan extension6. Furthermore, we established that fmo-2 also requires fmo-4 for its overexpression to extend lifespan4. Given these interconnected relationships, here we investigate whether deguelin and fmo-4 genetically interact in the context of longevity. Interestingly, our recent publication shows that fmo-4 extends lifespan downstream of the inhibition of mTORC1 (rsks-1 RNA interference) and has genetic ties to mitochondrial metabolism4. Considering these similarities between fmo-4 and deguelin, we hypothesized that deguelin may extend lifespan and promote healthspan through the induction of fmo-4, and we sought to investigate the downstream effectors involved in this process.
To address this hypothesis, we first examined the effects of deguelin on WT C. elegans lifespan. Our lab published that deguelin, in addition to multiple other compounds, extends lifespan of WT worms6, and so we aimed to validate these data using a range of concentrations. Our results confirm that deguelin extends WT lifespan in a dose-dependent manner (Figure 1A). To further investigate the quality of life during this extended lifespan, we assessed the healthspan of deguelin-treated worms. Our data show that deguelin not only extends lifespan but also promotes healthspan in middle-aged (day 8 of adulthood) WT worms (Figure 1B), indicating that the compound may improve overall health and functionality during aging.
To explore the molecular mechanisms underlying deguelin's effects, we focused on the potential involvement of fmo-4. We recently found that fmo-4 is required for mTOR pathway gene rsks-1-RNAi-mediated longevity, and that it extends lifespan by regulating calcium signaling between the ER and mitochondria4. This is interesting because deguelin is an inhibitor of both mTORC1 and mitochondrial complex I7, providing more evidence for a potential interaction between the two longevity interventions. Using a transcriptional fmo-4::mCherry reporter, we observed that deguelin treatment induces the expression of fmo-4 in C. elegans by ~2-fold. Fluorescent images and quantification reveal this significant increase in fmo-4 expression in deguelin-treated worms compared to the DMSO-treated control worms (Figure 1C-D). This induction suggests that fmo-4 is regulated by deguelin and may play a crucial role in mediating the longevity-promoting effects of deguelin. To confirm the requirement of fmo-4 in deguelin-mediated lifespan extension, we performed a lifespan assay using fmo-4 knockout (KO) worms. Our results demonstrate that the lifespan-extending effect of deguelin is abolished in fmo-4 KO worms (Figure 1E), indicating that fmo-4 is indeed necessary for and downstream of deguelin to extend lifespan in C. elegans. Similarly, we found that fmo-4 is also required for deguelin to promote healthspan in day 8 adult worms (Figure 1F), further supporting the critical role of fmo-4 in mediating the beneficial effects of deguelin on aging.
To gain insight into the downstream mechanisms of deguelin-mediated longevity, we investigated the involvement of mcu-1, a downstream effector of fmo-4-mediated longevity4,9. mcu-1 is a mitochondrial calcium uniporter that has been implicated in aging, stress response, and calcium regulation4,9. Since fmo-4 is required for the longevity and healthspan effect seen with deguelin treatment, and since mcu-1 acts downstream of fmo-44, we hypothesized that mcu-1 would also be required for the benefits of deguelin treatment. Our data show that mcu-1 is required for deguelin-mediated longevity (Figure 1G), suggesting that the effects of deguelin on lifespan extension may involve modulation of mitochondrial calcium homeostasis through the fmo-4/mcu-1 pathway.
While our findings provide compelling evidence for the role of deguelin in promoting longevity and healthspan in C. elegans through the induction of fmo-4, there are several limitations to consider. First, the exact mechanism by which deguelin induces fmo-4 expression remains to be elucidated. It is possible that deguelin, fmo-2, and fmo-4 are working in the same genetic pathway to promote these health benefits but more work needs to be done to confirm this involvement. Additionally, further research is needed to determine whether the effects of deguelin on lifespan and healthspan are conserved in other organisms, including mammals. Finally, potential off-target effects of deguelin and long-term consequences of its administration should be carefully evaluated in future studies.
In summary, our data demonstrate that deguelin extends lifespan and promotes healthspan in C. elegans in an fmo-4-dependent manner. We find that deguelin induces the expression of fmo-4 and that both fmo-4 and its downstream effector mcu-1 are required for deguelin-mediated longevity. These findings contribute to our understanding of the molecular mechanisms underlying lifespan extension and highlight the potential of deguelin as a pro-longevity compound. Future directions for this research include investigating the upstream regulators of fmo-4 induction by deguelin, exploring the conservation of this pathway in higher organisms, and evaluating the potential of deguelin or related compounds as interventions to promote healthy aging in humans.
","references":[{"reference":"Guo J, Huang X, Dou L, Yan M, Shen T, Tang W, Li J. 2022. Aging and aging-related diseases: from molecular mechanisms to interventions and treatments. Signal Transduct Target Ther 7(1): 391.
","pubmedId":"36522308","doi":""},{"reference":"Kenyon CJ. 2010. The genetics of ageing. Nature 464(7288): 504-12.
","pubmedId":"20336132","doi":""},{"reference":"Leiser SF, Miller H, Rossner R, Fletcher M, Leonard A, Primitivo M, et al., Kaeberlein M. 2015. Cell nonautonomous activation of flavin-containing monooxygenase promotes longevity and health span. Science 350(6266): 1375-1378.
","pubmedId":"26586189","doi":""},{"reference":"Tuckowski AM, Beydoun S, Kitto ES, et al. fmo-4 promotes longevity and stress resistance via ER to mitochondria calcium regulation in C. elegans. Elife. Feb 14 2025; 13doi:10.7554/eLife.99971
","pubmedId":"","doi":""},{"reference":"Choi HS, Bhat A, Howington MB, Schaller ML, Cox RL, Huang S, et al., Leiser SF. 2023. FMO rewires metabolism to promote longevity through tryptophan and one carbon metabolism in C. elegans. Nat Commun 14(1): 562.
","pubmedId":"36732543","doi":""},{"reference":"Huang S, Cox RL, Tuckowski A, Beydoun S, Bhat A, Howington MB, et al., Leiser SF. 2024. Fmo induction as a tool to screen for pro-longevity drugs. Geroscience 46(5): 4689-4706.
","pubmedId":"38787463","doi":""},{"reference":"Carpenter EL, Chagani S, Nelson D, Cassidy PB, Laws M, Ganguli-Indra G, Indra AK. 2019. Mitochondrial complex I inhibitor deguelin induces metabolic reprogramming and sensitizes vemurafenib-resistant BRAF(V600E) mutation bearing metastatic melanoma cells. Mol Carcinog 58(9): 1680-1690.
","pubmedId":"31211467","doi":""},{"reference":"Xu H, Li X, Ding W, Zeng X, Kong H, Wang H, Xie W. 2015. Deguelin induces the apoptosis of lung cancer cells through regulating a ROS driven Akt pathway. Cancer Cell Int 15: 25.
","pubmedId":"25741219","doi":""},{"reference":"Marchi S, Pinton P. 2014. The mitochondrial calcium uniporter complex: molecular components, structure and physiopathological implications. J Physiol 592(5): 829-39.
","pubmedId":"24366263","doi":""}],"suggestedReviewer":{"name":"Kristopher Burkewitz kristopher.burkewitz@Vanderbilt.Edu
Mark McCormick mmccormick@salud.unm.edu
Jeremy Van Raamsdonk jeremy.vanraamsdonk@mcgill.ca
","WBId":""},"title":"Deguelin promotes longevity and healthspan through C. elegans fmo-4
","reviews":[{"reviewer":{"displayName":"Mark McCormick"},"openAcknowledgement":false,"status":{"submitted":true}}]},{"id":"9970d48c-4765-4c8b-9424-3012bc61712d","decisionLetter":null,"decision":"edit","submitted":true,"abstract":"There are multiple approaches to longevity interventions in Caenorhabditis elegans, including genetic factors that are necessary or sufficient for lifespan extension and pharmacological agents that modify physiology to extend lifespan. Many pharmacological interventions act through known genetic pathways to promote longevity. Here, we show that the mitochondrial complex I inhibitor, deguelin, promotes lifespan extension and healthspan in an fmo-4-dependent manner. Our results confirm that deguelin increases lifespan and indicate that deguelin induces and requires multiple FMO enzymes to extend lifespan in C. elegans, suggesting these enzymes may promote longevity in a coordinated fashion.
","acknowledgements":"This work was supported by grants from NIH. AMT was supported by NIH T32AG000114 and the University of Michigan Rackham Research Grant. SFL was supported by R01AG075061 and the Glenn Foundation for Medical Research.
","authors":[{"affiliations":["University of Michigan"],"credit":["conceptualization","formalAnalysis","investigation","methodology","writing_originalDraft","writing_reviewEditing"],"email":"atuckow@umich.edu","firstName":"Angela M","lastName":"Tuckowski","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9125-4780"},{"affiliations":["Kansas State University"],"credit":["conceptualization","writing_reviewEditing"],"email":"shijiaoh@ksu.edu","firstName":"Shijiao","lastName":"Huang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["methodology","writing_reviewEditing"],"email":"chamberskelly5698@gmail.com","firstName":"Kelly","lastName":"Chambers","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["methodology","writing_reviewEditing"],"email":"bubrando@umich.edu","firstName":"Brandon","lastName":"Buscher","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["writing_reviewEditing","supervision","resources"],"email":"leiser@umich.edu","firstName":"Scott F","lastName":"Leiser","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"comments":"","dataTable":null,"disclaimer":true,"funding":"This work was supported by grants from NIH. AMT was supported by NIH T32AG000114, NIH 5T32GM007315, and the University of Michigan Rackham Research Grant. SFL was supported by R01AG075061 and the Glenn Foundation for Medical Research.
","image":{"name":"Figure micropub Tuckowski.jpg","url":"https://portal.micropublication.org/uploads/a7dd596a42c4239875c87f451fbdf812.jpg"},"imageCaption":"(A) Lifespan assessment of wild-type (WT) worms exposed to DMSO control, 1 µM deguelin, 5 µM deguelin, or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis). (B) Healthspan analysis of wild-type (WT) worms exposed to DMSO control or 10 µM deguelin thrashing in a drop of M9 solution for 30 seconds on days 1 and 8 of adulthood (n = ~10 worms per condition, three replicate experiments). (C) Fluorescence intensity of fmo-4p::mCherry transcriptional reporter worms exposed to DMSO control or 10 µM deguelin (n = ~20 worms per condition, three replicate experiments), quantified in (D). (E) Lifespan assessment of wild-type (WT) and fmo-4 knockout (KO) worms exposed to DMSO control or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis and significant interactions between the condition of interest and genotype was determined at p < 0.01 using Cox regression analysis). (F) Healthspan analysis of wild-type (WT) and fmo-4 knockout (KO) worms exposed to DMSO control or 10 µM deguelin thrashing in a drop of M9 solution for 30 seconds on days 1 and 8 of adulthood (n = ~10 worms per condition, three replicate experiments). (G) Lifespan assessment of wild-type (WT) and mcu-1 knockout (KO) worms exposed to DMSO control or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis and significant interactions between condition of interest and genotype was determined at p < 0.01 using Cox regression analysis). For healthspan and imaging experiments, * denotes significant change at p < 0.05 using unpaired two-tailed t test. NS = not significant. All replicate data can be found in the Source Data files.
","imageTitle":"Deguelin requires fmo-4 to promote lifespan extension and improve healthspan in C. elegans.
","laboratory":{"name":"","WBId":""},"methods":"Strains and Maintenance
Standard C. elegans cultivation procedures were used as previously described3. Worm strains were maintained on solid nematode growth medium (NGM) using E. coli OP50 throughout life. Worms were transferred using a platinum wire. All worm strains were maintained at 20°C.
Lifespan Assays
Gravid adult worms were placed on NGM plates seeded with E. coli OP50 for three hours. Then the adults were removed and eggs were allowed to hatch and develop to day 1 adulthood at 20°C. Adult worms were transferred to NGM plates containing 25 µg/mL carbenicillin, floxuridine (FUdR), and either dimethylsulfoxide (DMSO), 1 µM deguelin (Sigma, D0817), 5 µM deguelin, or 10 µM deguelin. Additionally, these plates were seeded with 200 µL of paraformaldehyde (PFA) killed E. coli OP50 at a concentration of 3x. Approximately 70 worms were transferred to fresh plates on day 1, day 2, day 4, and day 6 of adulthood. Two plates per strain per condition were tested per replicate experiment. Experimental animals were scored every 2-3 days and considered dead when they did not move in response to prodding under a dissection microscope. Worms that crawled off the plate were not considered, but ruptured worms were considered as previously described3. Three replicates were performed for each lifespan assay.
Thrashing Assay
Worms were synchronized by placing 10 gravid adult worms on NGM plates seeded with E. coli OP50 and allowing them to lay eggs for 2 hours at 20°C. The gravid adults were removed and the eggs were allowed to hatch and develop at 20°C until larval stage 2 (L2). At this stage, the L2 worms were transferred to NGM plates containing either DMSO or 10 µM deguelin, seeded with paraformaldehyde killed E. coli OP50. On day 1 adulthood, worms were placed in a drop of M9 solution, as previously described. The body bends were counted at maximum rate for 30 seconds. Thrashing was assayed on day 1 and day 8 of adulthood. The worms that were not used for the day 1 assay were transferred to fresh plates containing either DMSO or deguelin two times until they were ready to be assayed. Three replicates were performed. Data were analyzed in GraphPad Prism using unpaired two-tailed t tests with Welch’s correction.
fmo-4 Induction on Deguelin
Gravid fmo-4p::mCherry transcriptional reporter adult animals were placed on NGM plates seeded with E. coli OP50. After 3 hours, adults were removed and the eggs were allowed to develop at 20°C until they reached larval stage 4 (L4). Then 30 of the L4 worms were transferred to NGM plates containing either DMSO or 10 µM deguelin and seeded with paraformaldehyde (PFA) killed E. coli OP50. The worms were incubated for 24 hours at 20°C. Then ~20 worms per condition were picked off these plates and added to unseeded NGM plates, anesthetized in 0.5 M sodium azide (Sigma), and imaged at 6.3x magnification with the LASx software and Leica scope using the mCherry fluorescence channel. Three replicates were performed. Each worm was measured for fluorescence in ImageJ. Data were analyzed in GraphPad Prism using t tests.
Statistical Analyses
Log-rank test was used to derive p-value for lifespan assays using p < 0.05 cut-off threshold compared to DMSO or wild-type controls. Cox regression was also used to assess interactions between genotype and condition for lifespans using p < 0.01 cut-off threshold compared to controls. Supplemental Data 1 provide the results of the Log-rank test and Cox regression analyses, which were run in RStudio.
Strain Name | Genotype | Source |
WT or N2 | WT | CGC |
RB562 (fmo-4 knockout) | fmo-4(ok294) | CGC |
CZ19982 (mcu-1 knockout) | mcu-1(ju1154) | CGC |
fmo-4 transcriptional reporter | fmo-4p::mCherry | Suny Bioscience |
Reagent | Source | Catalog # |
Deguelin | Sigma | D0817-5MG |
DMSO | Fisher Scientific | BP213-100 |
Sodium Azide | Sigma | S2002-5G |
Aging is a complex biological process that affects all living organisms, leading to a decline in physiological functions and an increased susceptibility to diseases1. Studying the aging process is crucial because it allows for a better understanding of how to prevent or delay the onset of multiple age-related chronic diseases simultaneously. The nematode Caenorhabditis elegans (C. elegans) has emerged as a powerful model organism for studying aging due to its short lifespan, well-characterized genetics, and conserved aging pathways2. Recent research has focused on identifying longevity interventions in C. elegans, including genetic factors and pharmacological agents that can extend lifespan and improve healthspan, potentially offering insights into interventions that could promote healthy aging in humans.
A gene family of particular interest are the flavin-containing monooxygenase (fmo) genes. From this family, fmo-2 and fmo-4 are both sufficient and necessary for lifespan extension in C. elegans3,4. These genes play crucial roles in altering cellular metabolism, including one carbon metabolism5 and calcium regulation between the endoplasmic reticulum and mitochondria4, contributing to the longevity phenotype. Pharmacologically, compounds like deguelin, a natural rotenoid isolated from plants of the Leguminosae family, have gained attention for their potential to extend lifespan through the manipulation or inhibition of cellular processes6-8. For instance, deguelin treatment has been successfully tested as an anti-tumor agent in human cells lines, inhibits mitochondrial complex I, inhibits mammalian target of rapamycin complex I (mTORC1), and extends lifespan in wild-type (WT) C. elegans6-8.
Our lab previously demonstrated that these interventions can act in the same genetic pathway to extend lifespan. Specifically, we have shown that deguelin requires fmo-2 to promote longevity in C. elegans, suggesting that fmo-2 acts downstream of deguelin-mediated lifespan extension6. Furthermore, we established that fmo-2 also requires fmo-4 for its overexpression to extend lifespan4. Given these interconnected relationships, here we investigate whether deguelin and fmo-4 genetically interact in the context of longevity. Interestingly, our recent publication shows that fmo-4 extends lifespan downstream of the inhibition of mTORC1 (rsks-1 RNA interference) and has genetic ties to mitochondrial metabolism4. Considering these similarities between fmo-4 and deguelin, we hypothesized that deguelin may extend lifespan and promote healthspan through the induction of fmo-4, and we sought to investigate the downstream effectors involved in this process.
To address this hypothesis, we first examined the effects of deguelin on WT C. elegans lifespan. Our lab published that deguelin, in addition to multiple other compounds, extends lifespan of WT worms6, and so we aimed to validate these data using a range of concentrations. Our results confirm that deguelin extends WT lifespan in a dose-dependent manner (Figure 1A). To further investigate the quality of life during this extended lifespan, we assessed the healthspan of deguelin-treated worms. Our data show that deguelin not only extends lifespan but also promotes healthspan in middle-aged (day 8 of adulthood) WT worms (Figure 1B), indicating that the compound may improve overall health and functionality during aging.
To explore the molecular mechanisms underlying deguelin's effects, we focused on the potential involvement of fmo-4. We recently found that fmo-4 is required for mTOR pathway gene rsks-1-RNAi-mediated longevity, and that it extends lifespan by regulating calcium signaling between the ER and mitochondria4. This is interesting because deguelin is an inhibitor of both mTORC1 and mitochondrial complex I7, providing more evidence for a potential interaction between the two longevity interventions. Using a transcriptional fmo-4::mCherry reporter, we observed that deguelin treatment induces the expression of fmo-4 in C. elegans by ~2-fold. Fluorescent images and quantification reveal this significant increase in fmo-4 expression in deguelin-treated worms compared to the DMSO-treated control worms (Figure 1C-D). This induction suggests that fmo-4 is regulated by deguelin and may play a crucial role in mediating the longevity-promoting effects of deguelin. To confirm the requirement of fmo-4 in deguelin-mediated lifespan extension, we performed a lifespan assay using fmo-4 knockout (KO) worms. Our results demonstrate that the lifespan-extending effect of deguelin is abolished in fmo-4 KO worms (Figure 1E), indicating that fmo-4 is indeed necessary for and downstream of deguelin to extend lifespan in C. elegans. Similarly, we found that fmo-4 is also required for deguelin to promote healthspan in day 8 adult worms (Figure 1F), further supporting the critical role of fmo-4 in mediating the beneficial effects of deguelin on aging.
To gain insight into the downstream mechanisms of deguelin-mediated longevity, we investigated the involvement of mcu-1, a downstream effector of fmo-4-mediated longevity4,9. mcu-1 is a mitochondrial calcium uniporter that has been implicated in aging, stress response, and calcium regulation4,9. Since fmo-4 is required for the longevity and healthspan effect seen with deguelin treatment, and since mcu-1 acts downstream of fmo-44, we hypothesized that mcu-1 would also be required for the benefits of deguelin treatment. Our data show that mcu-1 is required for deguelin-mediated longevity (Figure 1G), suggesting that the effects of deguelin on lifespan extension may involve modulation of mitochondrial calcium homeostasis through the fmo-4/mcu-1 pathway.
While our findings provide compelling evidence for the role of deguelin in promoting longevity and healthspan in C. elegans through the induction of fmo-4, there are several limitations to consider. First, the exact mechanism by which deguelin induces fmo-4 expression remains to be elucidated. It is possible that deguelin, fmo-2, and fmo-4 are working in the same genetic pathway to promote these health benefits but more work needs to be done to confirm this involvement. Additionally, further research is needed to determine whether the effects of deguelin on lifespan and healthspan are conserved in other organisms, including mammals. Finally, potential off-target effects of deguelin and long-term consequences of its administration should be carefully evaluated in future studies.
In summary, our data demonstrate that deguelin extends lifespan and promotes healthspan in C. elegans in an fmo-4-dependent manner. We find that deguelin induces the expression of fmo-4 and that both fmo-4 and its downstream effector mcu-1 are required for deguelin-mediated longevity. These findings contribute to our understanding of the molecular mechanisms underlying lifespan extension and highlight the potential of deguelin as a pro-longevity compound. Future directions for this research include investigating the upstream regulators of fmo-4 induction by deguelin, exploring the conservation of this pathway in higher organisms, and evaluating the potential of deguelin or related compounds as interventions to promote healthy aging in humans.
","references":[{"reference":"Guo J, Huang X, Dou L, Yan M, Shen T, Tang W, Li J. 2022. Aging and aging-related diseases: from molecular mechanisms to interventions and treatments. Signal Transduct Target Ther 7(1): 391.
","pubmedId":"36522308","doi":""},{"reference":"Kenyon CJ. 2010. The genetics of ageing. Nature 464(7288): 504-12.
","pubmedId":"20336132","doi":""},{"reference":"Leiser SF, Miller H, Rossner R, Fletcher M, Leonard A, Primitivo M, et al., Kaeberlein M. 2015. Cell nonautonomous activation of flavin-containing monooxygenase promotes longevity and health span. Science 350(6266): 1375-1378.
","pubmedId":"26586189","doi":""},{"reference":"Tuckowski AM, Beydoun S, Kitto ES, et al. fmo-4 promotes longevity and stress resistance via ER to mitochondria calcium regulation in C. elegans. Elife. Feb 14 2025; 13 doi:10.7554/eLife.99971
","pubmedId":"","doi":""},{"reference":"Choi HS, Bhat A, Howington MB, Schaller ML, Cox RL, Huang S, et al., Leiser SF. 2023. FMO rewires metabolism to promote longevity through tryptophan and one carbon metabolism in C. elegans. Nat Commun 14(1): 562.
","pubmedId":"36732543","doi":""},{"reference":"Huang S, Cox RL, Tuckowski A, Beydoun S, Bhat A, Howington MB, et al., Leiser SF. 2024. Fmo induction as a tool to screen for pro-longevity drugs. Geroscience 46(5): 4689-4706.
","pubmedId":"38787463","doi":""},{"reference":"Carpenter EL, Chagani S, Nelson D, Cassidy PB, Laws M, Ganguli-Indra G, Indra AK. 2019. Mitochondrial complex I inhibitor deguelin induces metabolic reprogramming and sensitizes vemurafenib-resistant BRAF(V600E) mutation bearing metastatic melanoma cells. Mol Carcinog 58(9): 1680-1690.
","pubmedId":"31211467","doi":""},{"reference":"Xu H, Li X, Ding W, Zeng X, Kong H, Wang H, Xie W. 2015. Deguelin induces the apoptosis of lung cancer cells through regulating a ROS driven Akt pathway. Cancer Cell Int 15: 25.
","pubmedId":"25741219","doi":""},{"reference":"Marchi S, Pinton P. 2014. The mitochondrial calcium uniporter complex: molecular components, structure and physiopathological implications. J Physiol 592(5): 829-39.
","pubmedId":"24366263","doi":""}],"suggestedReviewer":{"name":"Kristopher Burkewitz kristopher.burkewitz@Vanderbilt.Edu
Mark McCormick mmccormick@salud.unm.edu
Jeremy Van Raamsdonk jeremy.vanraamsdonk@mcgill.ca
","WBId":""},"title":"Deguelin promotes longevity and healthspan through C. elegans fmo-4
","reviews":[]},{"id":"5226c821-5c4d-4cf5-8e9d-5367b65b57ed","decisionLetter":"Dear Angela M Tuckowski,
Thank you for submitting a revised version of the manuscript \"Deguelin promotes longevity and healthspan through C. elegans fmo-4\" to microPublication Biology.
Before we can move further, there are some very minor additional issues that need to be addressed:
Below are the comments from the reviewer
This clear and straightforward paper by Tuckowski et al. describes effects of the mitochondrial complex I inhibitor deguelin on lifespan and healthspan in the nematode Caenorhabditis elegans.
The authors demonstrate a dose-dependent increase in survival upon deguelin treatment, with a maximum effect at a dose of 10uM. They use thrashing as a measure of fitness and show that in Day 8 worms deguelin treated worms have a significantly higher thrashing rate than untreated controls, making the Day 8 deguelin treated worms more similar to younger untreated worms.
The authors show that deguelin treatment can induce an fmo-4p::mCherry transcriptional reporter, and that fmo-4 KO prevents the increased lifespan and increased Day 8 thrashing seen in treated animals.
Next the authors look at mcu-1, a mitochondrial calcium uniporter that has been previously implicated in aging and previously shown to be downstream of fmo-4. They find that mcu-1 KO prevents lifespan extension by deguelin treatment at the doses tested.
Overall this is a very interesting addition to the field of aging research broadly, and even more so to those who use C. elegans as a model system. The manuscript and figures including the methods are clear and well written.
Specific comments.
1. In both x-axis labels of Figure 1F I believe that \"fmo-4\" should be italicized.
2. \"A gene family of particular interest are the flavin-containing monooxygenase (fmo) genes. From this family, fmo-2 and fmo-4 are both sufficient and necessary for lifespan extension in C. elegans3,4. \" Here the footnotes for references 3 and 4 have been inadvertently italicized probably as they follow \"C. elegans\".
We kindly ask you to address each point and summarize your changes in line with the request in the 'Comments to Editor' section on the platform. In order to expedite the processing of your revised manuscript, please be as specific as possible in your responses.
The microPublication Editorial Team
","decision":"revise","submitted":true,"abstract":"There are multiple approaches to longevity interventions in Caenorhabditis elegans, including genetic factors that are necessary or sufficient for lifespan extension and pharmacological agents that modify physiology to extend lifespan. Many pharmacological interventions act through known genetic pathways to promote longevity. Here, we show that the mitochondrial complex I inhibitor, deguelin, promotes lifespan extension and healthspan in an fmo-4-dependent manner. Our results confirm that deguelin increases lifespan and indicate that deguelin induces and requires multiple FMO enzymes to extend lifespan in C. elegans, suggesting these enzymes may promote longevity in a coordinated fashion.
","acknowledgements":"This work was supported by grants from NIH. AMT was supported by NIH T32AG000114 and the University of Michigan Rackham Research Grant. SFL was supported by R01AG075061 and the Glenn Foundation for Medical Research.
","authors":[{"affiliations":["University of Michigan"],"credit":["conceptualization","formalAnalysis","investigation","methodology","writing_originalDraft","writing_reviewEditing"],"email":"atuckow@umich.edu","firstName":"Angela M","lastName":"Tuckowski","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9125-4780"},{"affiliations":["Kansas State University"],"credit":["conceptualization","writing_reviewEditing"],"email":"shijiaoh@ksu.edu","firstName":"Shijiao","lastName":"Huang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["methodology","writing_reviewEditing"],"email":"chamberskelly5698@gmail.com","firstName":"Kelly","lastName":"Chambers","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["methodology","writing_reviewEditing"],"email":"bubrando@umich.edu","firstName":"Brandon","lastName":"Buscher","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["writing_reviewEditing","supervision","resources"],"email":"leiser@umich.edu","firstName":"Scott F","lastName":"Leiser","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"comments":"","dataTable":null,"disclaimer":true,"funding":"This work was supported by grants from NIH. AMT was supported by NIH T32AG000114, NIH 5T32GM007315, and the University of Michigan Rackham Research Grant. SFL was supported by R01AG075061 and the Glenn Foundation for Medical Research.
","image":{"name":"Figure micropub Tuckowski.jpg","url":"https://portal.micropublication.org/uploads/a7dd596a42c4239875c87f451fbdf812.jpg"},"imageCaption":"(A) Lifespan assessment of wild-type (WT) worms exposed to DMSO control, 1 µM deguelin, 5 µM deguelin, or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis). (B) Healthspan analysis of wild-type (WT) worms exposed to DMSO control or 10 µM deguelin thrashing in a drop of M9 solution for 30 seconds on days 1 and 8 of adulthood (n = ~10 worms per condition, three replicate experiments). (C) Fluorescence intensity of fmo-4p::mCherry transcriptional reporter worms exposed to DMSO control or 10 µM deguelin (n = ~20 worms per condition, three replicate experiments), quantified in (D). (E) Lifespan assessment of wild-type (WT) and fmo-4 knockout (KO) worms exposed to DMSO control or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis and significant interactions between the condition of interest and genotype was determined at p < 0.01 using Cox regression analysis). (F) Healthspan analysis of wild-type (WT) and fmo-4 knockout (KO) worms exposed to DMSO control or 10 µM deguelin thrashing in a drop of M9 solution for 30 seconds on days 1 and 8 of adulthood (n = ~10 worms per condition, three replicate experiments). (G) Lifespan assessment of wild-type (WT) and mcu-1 knockout (KO) worms exposed to DMSO control or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis and significant interactions between condition of interest and genotype was determined at p < 0.01 using Cox regression analysis). For healthspan and imaging experiments, * denotes significant change at p < 0.05 using unpaired two-tailed t test. NS = not significant. All replicate data can be found in the Source Data files.
","imageTitle":"Deguelin requires fmo-4 to promote lifespan extension and improve healthspan in C. elegans.
","laboratory":{"name":"","WBId":""},"methods":"Strains and Maintenance
Standard C. elegans cultivation procedures were used as previously described3. Worm strains were maintained on solid nematode growth medium (NGM) using E. coli OP50 throughout life. Worms were transferred using a platinum wire. All worm strains were maintained at 20°C.
Lifespan Assays
Gravid adult worms were placed on NGM plates seeded with E. coli OP50 for three hours. Then the adults were removed and eggs were allowed to hatch and develop to day 1 adulthood at 20°C. Adult worms were transferred to NGM plates containing 25 µg/mL carbenicillin, floxuridine (FUdR), and either dimethylsulfoxide (DMSO), 1 µM deguelin (Sigma, D0817), 5 µM deguelin, or 10 µM deguelin. Additionally, these plates were seeded with 200 µL of paraformaldehyde (PFA) killed E. coli OP50 at a concentration of 3x. Approximately 70 worms were transferred to fresh plates on day 1, day 2, day 4, and day 6 of adulthood. Two plates per strain per condition were tested per replicate experiment. Experimental animals were scored every 2-3 days and considered dead when they did not move in response to prodding under a dissection microscope. Worms that crawled off the plate were not considered, but ruptured worms were considered as previously described3. Three replicates were performed for each lifespan assay.
Thrashing Assay
Worms were synchronized by placing 10 gravid adult worms on NGM plates seeded with E. coli OP50 and allowing them to lay eggs for 2 hours at 20°C. The gravid adults were removed and the eggs were allowed to hatch and develop at 20°C until larval stage 2 (L2). At this stage, the L2 worms were transferred to NGM plates containing either DMSO or 10 µM deguelin, seeded with paraformaldehyde killed E. coli OP50. On day 1 adulthood, worms were placed in a drop of M9 solution, as previously described. The body bends were counted at maximum rate for 30 seconds. Thrashing was assayed on day 1 and day 8 of adulthood. The worms that were not used for the day 1 assay were transferred to fresh plates containing either DMSO or deguelin two times until they were ready to be assayed. Three replicates were performed. Data were analyzed in GraphPad Prism using unpaired two-tailed t tests with Welch’s correction.
fmo-4 Induction on Deguelin
Gravid fmo-4p::mCherry transcriptional reporter adult animals were placed on NGM plates seeded with E. coli OP50. After 3 hours, adults were removed and the eggs were allowed to develop at 20°C until they reached larval stage 4 (L4). Then 30 of the L4 worms were transferred to NGM plates containing either DMSO or 10 µM deguelin and seeded with paraformaldehyde (PFA) killed E. coli OP50. The worms were incubated for 24 hours at 20°C. Then ~20 worms per condition were picked off these plates and added to unseeded NGM plates, anesthetized in 0.5 M sodium azide (Sigma), and imaged at 6.3x magnification with the LASx software and Leica scope using the mCherry fluorescence channel. Three replicates were performed. Each worm was measured for fluorescence in ImageJ. Data were analyzed in GraphPad Prism using t tests.
Statistical Analyses
Log-rank test was used to derive p-value for lifespan assays using p < 0.05 cut-off threshold compared to DMSO or wild-type controls. Cox regression was also used to assess interactions between genotype and condition for lifespans using p < 0.01 cut-off threshold compared to controls. Supplemental Data 1 provide the results of the Log-rank test and Cox regression analyses, which were run in RStudio.
Strain Name | Genotype | Source |
WT or N2 | WT | CGC |
RB562 (fmo-4 knockout) | fmo-4(ok294) | CGC |
CZ19982 (mcu-1 knockout) | mcu-1(ju1154) | CGC |
fmo-4 transcriptional reporter | fmo-4p::mCherry | Suny Bioscience |
Reagent | Source | Catalog # |
Deguelin | Sigma | D0817-5MG |
DMSO | Fisher Scientific | BP213-100 |
Sodium Azide | Sigma | S2002-5G |
Aging is a complex biological process that affects all living organisms, leading to a decline in physiological functions and an increased susceptibility to diseases1. Studying the aging process is crucial because it allows for a better understanding of how to prevent or delay the onset of multiple age-related chronic diseases simultaneously. The nematode Caenorhabditis elegans (C. elegans) has emerged as a powerful model organism for studying aging due to its short lifespan, well-characterized genetics, and conserved aging pathways2. Recent research has focused on identifying longevity interventions in C. elegans, including genetic factors and pharmacological agents that can extend lifespan and improve healthspan, potentially offering insights into interventions that could promote healthy aging in humans.
A gene family of particular interest are the flavin-containing monooxygenase (fmo) genes. From this family, fmo-2 and fmo-4 are both sufficient and necessary for lifespan extension in C. elegans3,4. These genes play crucial roles in altering cellular metabolism, including one carbon metabolism5 and calcium regulation between the endoplasmic reticulum and mitochondria4, contributing to the longevity phenotype. Pharmacologically, compounds like deguelin, a natural rotenoid isolated from plants of the Leguminosae family, have gained attention for their potential to extend lifespan through the manipulation or inhibition of cellular processes6-8. For instance, deguelin treatment has been successfully tested as an anti-tumor agent in human cells lines, inhibits mitochondrial complex I, inhibits mammalian target of rapamycin complex I (mTORC1), and extends lifespan in wild-type (WT) C. elegans6-8.
Our lab previously demonstrated that these interventions can act in the same genetic pathway to extend lifespan. Specifically, we have shown that deguelin requires fmo-2 to promote longevity in C. elegans, suggesting that fmo-2 acts downstream of deguelin-mediated lifespan extension6. Furthermore, we established that fmo-2 also requires fmo-4 for its overexpression to extend lifespan4. Given these interconnected relationships, here we investigate whether deguelin and fmo-4 genetically interact in the context of longevity. Interestingly, our recent publication shows that fmo-4 extends lifespan downstream of the inhibition of mTORC1 (rsks-1 RNA interference) and has genetic ties to mitochondrial metabolism4. Considering these similarities between fmo-4 and deguelin, we hypothesized that deguelin may extend lifespan and promote healthspan through the induction of fmo-4, and we sought to investigate the downstream effectors involved in this process.
To address this hypothesis, we first examined the effects of deguelin on WT C. elegans lifespan. Our lab published that deguelin, in addition to multiple other compounds, extends lifespan of WT worms6, and so we aimed to validate these data using a range of concentrations. Our results confirm that deguelin extends WT lifespan in a dose-dependent manner (Figure 1A). To further investigate the quality of life during this extended lifespan, we assessed the healthspan of deguelin-treated worms. Our data show that deguelin not only extends lifespan but also promotes healthspan in middle-aged (day 8 of adulthood) WT worms (Figure 1B), indicating that the compound may improve overall health and functionality during aging.
To explore the molecular mechanisms underlying deguelin's effects, we focused on the potential involvement of fmo-4. We recently found that fmo-4 is required for mTOR pathway gene rsks-1-RNAi-mediated longevity, and that it extends lifespan by regulating calcium signaling between the ER and mitochondria4. This is interesting because deguelin is an inhibitor of both mTORC1 and mitochondrial complex I7, providing more evidence for a potential interaction between the two longevity interventions. Using a transcriptional fmo-4::mCherry reporter, we observed that deguelin treatment induces the expression of fmo-4 in C. elegans by ~2-fold. Fluorescent images and quantification reveal this significant increase in fmo-4 expression in deguelin-treated worms compared to the DMSO-treated control worms (Figure 1C-D). This induction suggests that fmo-4 is regulated by deguelin and may play a crucial role in mediating the longevity-promoting effects of deguelin. To confirm the requirement of fmo-4 in deguelin-mediated lifespan extension, we performed a lifespan assay using fmo-4 knockout (KO) worms. Our results demonstrate that the lifespan-extending effect of deguelin is abolished in fmo-4 KO worms (Figure 1E), indicating that fmo-4 is indeed necessary for and downstream of deguelin to extend lifespan in C. elegans. Similarly, we found that fmo-4 is also required for deguelin to promote healthspan in day 8 adult worms (Figure 1F), further supporting the critical role of fmo-4 in mediating the beneficial effects of deguelin on aging.
To gain insight into the downstream mechanisms of deguelin-mediated longevity, we investigated the involvement of mcu-1, a downstream effector of fmo-4-mediated longevity4,9. mcu-1 is a mitochondrial calcium uniporter that has been implicated in aging, stress response, and calcium regulation4,9. Since fmo-4 is required for the longevity and healthspan effect seen with deguelin treatment, and since mcu-1 acts downstream of fmo-44, we hypothesized that mcu-1 would also be required for the benefits of deguelin treatment. Our data show that mcu-1 is required for deguelin-mediated longevity (Figure 1G), suggesting that the effects of deguelin on lifespan extension may involve modulation of mitochondrial calcium homeostasis through the fmo-4/mcu-1 pathway.
While our findings provide compelling evidence for the role of deguelin in promoting longevity and healthspan in C. elegans through the induction of fmo-4, there are several limitations to consider. First, the exact mechanism by which deguelin induces fmo-4 expression remains to be elucidated. It is possible that deguelin, fmo-2, and fmo-4 are working in the same genetic pathway to promote these health benefits but more work needs to be done to confirm this involvement. Additionally, further research is needed to determine whether the effects of deguelin on lifespan and healthspan are conserved in other organisms, including mammals. Finally, potential off-target effects of deguelin and long-term consequences of its administration should be carefully evaluated in future studies.
In summary, our data demonstrate that deguelin extends lifespan and promotes healthspan in C. elegans in an fmo-4-dependent manner. We find that deguelin induces the expression of fmo-4 and that both fmo-4 and its downstream effector mcu-1 are required for deguelin-mediated longevity. These findings contribute to our understanding of the molecular mechanisms underlying lifespan extension and highlight the potential of deguelin as a pro-longevity compound. Future directions for this research include investigating the upstream regulators of fmo-4 induction by deguelin, exploring the conservation of this pathway in higher organisms, and evaluating the potential of deguelin or related compounds as interventions to promote healthy aging in humans.
","references":[{"reference":"Guo J, Huang X, Dou L, Yan M, Shen T, Tang W, Li J. 2022. Aging and aging-related diseases: from molecular mechanisms to interventions and treatments. Signal Transduct Target Ther 7(1): 391.
","pubmedId":"36522308","doi":""},{"reference":"Kenyon CJ. 2010. The genetics of ageing. Nature 464(7288): 504-12.
","pubmedId":"20336132","doi":""},{"reference":"Leiser SF, Miller H, Rossner R, Fletcher M, Leonard A, Primitivo M, et al., Kaeberlein M. 2015. Cell nonautonomous activation of flavin-containing monooxygenase promotes longevity and health span. Science 350(6266): 1375-1378.
","pubmedId":"26586189","doi":""},{"reference":"Tuckowski AM, Beydoun S, Kitto ES, Bhat A, Howington MB, Sridhar A, et al., Leiser. 2025. fmo-4 promotes longevity and stress resistance via ER to mitochondria calcium regulation in C. elegans. eLife 13: 10.7554/elife.99971.
","pubmedId":"","doi":"10.7554/eLife.99971"},{"reference":"Choi HS, Bhat A, Howington MB, Schaller ML, Cox RL, Huang S, et al., Leiser SF. 2023. FMO rewires metabolism to promote longevity through tryptophan and one carbon metabolism in C. elegans. Nat Commun 14(1): 562.
","pubmedId":"36732543","doi":""},{"reference":"Huang S, Cox RL, Tuckowski A, Beydoun S, Bhat A, Howington MB, et al., Leiser SF. 2024. Fmo induction as a tool to screen for pro-longevity drugs. Geroscience 46(5): 4689-4706.
","pubmedId":"38787463","doi":""},{"reference":"Carpenter EL, Chagani S, Nelson D, Cassidy PB, Laws M, Ganguli-Indra G, Indra AK. 2019. Mitochondrial complex I inhibitor deguelin induces metabolic reprogramming and sensitizes vemurafenib-resistant BRAF(V600E) mutation bearing metastatic melanoma cells. Mol Carcinog 58(9): 1680-1690.
","pubmedId":"31211467","doi":""},{"reference":"Xu H, Li X, Ding W, Zeng X, Kong H, Wang H, Xie W. 2015. Deguelin induces the apoptosis of lung cancer cells through regulating a ROS driven Akt pathway. Cancer Cell Int 15: 25.
","pubmedId":"25741219","doi":""},{"reference":"Marchi S, Pinton P. 2014. The mitochondrial calcium uniporter complex: molecular components, structure and physiopathological implications. J Physiol 592(5): 829-39.
","pubmedId":"24366263","doi":""}],"suggestedReviewer":{"name":"Kristopher Burkewitz kristopher.burkewitz@Vanderbilt.Edu
Mark McCormick mmccormick@salud.unm.edu
Jeremy Van Raamsdonk jeremy.vanraamsdonk@mcgill.ca
","WBId":""},"title":"Deguelin promotes longevity and healthspan through C. elegans fmo-4
","reviews":[]},{"id":"a2397834-d81c-4490-8928-70a56b0c79d6","decisionLetter":"\n\n Dear Angela M Tuckowski,\n
\n\n We are happy to let you know that your article has been accepted for publication. Congratulations!\n
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\n ","decision":"accept","submitted":true,"abstract":"There are multiple approaches to longevity interventions in Caenorhabditis elegans, including genetic factors that are necessary or sufficient for lifespan extension and pharmacological agents that modify physiology to extend lifespan. Many pharmacological interventions act through known genetic pathways to promote longevity. Here, we show that the mitochondrial complex I inhibitor, deguelin, promotes lifespan extension and healthspan in an fmo-4-dependent manner. Our results confirm that deguelin increases lifespan and indicate that deguelin induces and requires multiple FMO enzymes to extend lifespan in C. elegans, suggesting these enzymes may promote longevity in a coordinated fashion.
","acknowledgements":"","authors":[{"affiliations":["University of Michigan"],"credit":["conceptualization","formalAnalysis","investigation","methodology","writing_originalDraft","writing_reviewEditing"],"email":"atuckow@umich.edu","firstName":"Angela M","lastName":"Tuckowski","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9125-4780"},{"affiliations":["Kansas State University"],"credit":["conceptualization","writing_reviewEditing"],"email":"shijiaoh@ksu.edu","firstName":"Shijiao","lastName":"Huang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["methodology","writing_reviewEditing"],"email":"chamberskelly5698@gmail.com","firstName":"Kelly","lastName":"Chambers","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["methodology","writing_reviewEditing"],"email":"bubrando@umich.edu","firstName":"Brandon","lastName":"Buscher","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["writing_reviewEditing","supervision","resources"],"email":"leiser@umich.edu","firstName":"Scott F","lastName":"Leiser","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"comments":"We thank the reviewer for their thoughtful and constructive comments on our manuscript \"Deguelin promotes longevity and healthspan through C. elegans fmo-4.\" We appreciate the time and effort they have devoted to providing this feedback. We have considered the comments and have addressed them in a revised manuscript as detailed below.
Comment 1: In both x-axis labels of Figure 1F I believe that \"fmo-4\" should be italicized.
Response 1: We thank the reviewer for this helpful revision. We have made the change as suggested in the Figure 1F x-axis labels.
Comment 2: \"A gene family of particular interest are the flavin-containing monooxygenase (fmo) genes. From this family fmo-2 and fmo-4 are both sufficient and necessary for lifespan extension in C. elegans3,4.\" Here the footnotes for references 3 and 4 have been inadvertently italicized probably as they follow \"C. elegans.\"
Response 2: Thank you for identifying this. We have made the change as suggested. Additionally, we made sure to look through the rest of the manuscript for similar issues and fixed the italicized footnotes here: \"...inhibits mammalian target of rapamycin complex I (mTORC1), and extends lifespan in wild-type (WT) C. elegans6-8.\"
","dataTable":null,"disclaimer":true,"funding":"This work was supported by grants from NIH. AMT was supported by NIH T32AG000114, NIH 5T32GM007315, and the University of Michigan Rackham Research Grant. SFL was supported by R01AG075061 and the Glenn Foundation for Medical Research.
","image":{"name":"Figure Revised microPub Tuckowski.jpg","url":"https://portal.micropublication.org/uploads/1659665096a38cdd38c009e8c4028853.jpg"},"imageCaption":"(A) Lifespan assessment of wild-type (WT) worms exposed to DMSO control, 1 µM deguelin, 5 µM deguelin, or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis). (B) Healthspan analysis of wild-type (WT) worms exposed to DMSO control or 10 µM deguelin thrashing in a drop of M9 solution for 30 seconds on days 1 and 8 of adulthood (n = ~10 worms per condition, three replicate experiments). (C) Fluorescence intensity of fmo-4p::mCherry transcriptional reporter worms exposed to DMSO control or 10 µM deguelin (n = ~20 worms per condition, three replicate experiments), quantified in (D). (E) Lifespan assessment of wild-type (WT) and fmo-4 knockout (KO) worms exposed to DMSO control or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis and significant interactions between the condition of interest and genotype was determined at p < 0.01 using Cox regression analysis). (F) Healthspan analysis of wild-type (WT) and fmo-4 knockout (KO) worms exposed to DMSO control or 10 µM deguelin thrashing in a drop of M9 solution for 30 seconds on days 1 and 8 of adulthood (n = ~10 worms per condition, three replicate experiments). (G) Lifespan assessment of wild-type (WT) and mcu-1 knockout (KO) worms exposed to DMSO control or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis and significant interactions between condition of interest and genotype was determined at p < 0.01 using Cox regression analysis). For healthspan and imaging experiments, * denotes significant change at p < 0.05 using unpaired two-tailed t test. NS = not significant. All replicate data can be found in the Source Data files.
","imageTitle":"Deguelin requires fmo-4 to promote lifespan extension and improve healthspan in C. elegans.
","laboratory":{"name":"","WBId":""},"methods":"Strains and Maintenance
Standard C. elegans cultivation procedures were used as previously described3. Worm strains were maintained on solid nematode growth medium (NGM) using E. coli OP50 throughout life. Worms were transferred using a platinum wire. All worm strains were maintained at 20°C.
Lifespan Assays
Gravid adult worms were placed on NGM plates seeded with E. coli OP50 for three hours. Then the adults were removed and eggs were allowed to hatch and develop to day 1 adulthood at 20°C. Adult worms were transferred to NGM plates containing 25 µg/mL carbenicillin, floxuridine (FUdR), and either dimethylsulfoxide (DMSO), 1 µM deguelin (Sigma, D0817), 5 µM deguelin, or 10 µM deguelin. Additionally, these plates were seeded with 200 µL of paraformaldehyde (PFA) killed E. coli OP50 at a concentration of 3x. Approximately 70 worms were transferred to fresh plates on day 1, day 2, day 4, and day 6 of adulthood. Two plates per strain per condition were tested per replicate experiment. Experimental animals were scored every 2-3 days and considered dead when they did not move in response to prodding under a dissection microscope. Worms that crawled off the plate were not considered, but ruptured worms were considered as previously described3. Three replicates were performed for each lifespan assay.
Thrashing Assay
Worms were synchronized by placing 10 gravid adult worms on NGM plates seeded with E. coli OP50 and allowing them to lay eggs for 2 hours at 20°C. The gravid adults were removed and the eggs were allowed to hatch and develop at 20°C until larval stage 2 (L2). At this stage, the L2 worms were transferred to NGM plates containing either DMSO or 10 µM deguelin, seeded with paraformaldehyde killed E. coli OP50. On day 1 adulthood, worms were placed in a drop of M9 solution, as previously described. The body bends were counted at maximum rate for 30 seconds. Thrashing was assayed on day 1 and day 8 of adulthood. The worms that were not used for the day 1 assay were transferred to fresh plates containing either DMSO or deguelin two times until they were ready to be assayed. Three replicates were performed. Data were analyzed in GraphPad Prism using unpaired two-tailed t tests with Welch's correction.
fmo-4 Induction on Deguelin
Gravid fmo-4p::mCherry transcriptional reporter adult animals were placed on NGM plates seeded with E. coli OP50. After 3 hours, adults were removed and the eggs were allowed to develop at 20°C until they reached larval stage 4 (L4). Then 30 of the L4 worms were transferred to NGM plates containing either DMSO or 10 µM deguelin and seeded with paraformaldehyde (PFA) killed E. coli OP50. The worms were incubated for 24 hours at 20°C. Then ~20 worms per condition were picked off these plates and added to unseeded NGM plates, anesthetized in 0.5 M sodium azide (Sigma), and imaged at 6.3x magnification with the LASx software and Leica scope using the mCherry fluorescence channel. Three replicates were performed. Each worm was measured for fluorescence in ImageJ. Data were analyzed in GraphPad Prism using t tests.
Statistical Analyses
Log-rank test was used to derive p-value for lifespan assays using p < 0.05 cut-off threshold compared to DMSO or wild-type controls. Cox regression was also used to assess interactions between genotype and condition for lifespans using p < 0.01 cut-off threshold compared to controls. Supplemental Data 1 provide the results of the Log-rank test and Cox regression analyses, which were run in RStudio.
Strain Name | Genotype | Source |
WT or N2 | WT | CGC |
CGC | ||
CGC | ||
fmo-4 transcriptional reporter | fmo-4p::mCherry | Suny Bioscience |
Reagent | Source | Catalog # |
Deguelin | Sigma | D0817-5MG |
DMSO | Fisher Scientific | BP213-100 |
Sodium Azide | Sigma | S2002-5G |
Aging is a complex biological process that affects all living organisms, leading to a decline in physiological functions and an increased susceptibility to diseases1. Studying the aging process is crucial because it allows for a better understanding of how to prevent or delay the onset of multiple age-related chronic diseases simultaneously. The nematode Caenorhabditis elegans (C. elegans) has emerged as a powerful model organism for studying aging due to its short lifespan, well-characterized genetics, and conserved aging pathways2. Recent research has focused on identifying longevity interventions in C. elegans, including genetic factors and pharmacological agents that can extend lifespan and improve healthspan, potentially offering insights into interventions that could promote healthy aging in humans.
A gene family of particular interest are the flavin-containing monooxygenase (fmo) genes. From this family, fmo-2 and fmo-4 are both sufficient and necessary for lifespan extension in C. elegans3,4. These genes play crucial roles in altering cellular metabolism, including one carbon metabolism5 and calcium regulation between the endoplasmic reticulum and mitochondria4, contributing to the longevity phenotype. Pharmacologically, compounds like deguelin, a natural rotenoid isolated from plants of the Leguminosae family, have gained attention for their potential to extend lifespan through the manipulation or inhibition of cellular processes6-8. For instance, deguelin treatment has been successfully tested as an anti-tumor agent in human cells lines, inhibits mitochondrial complex I, inhibits mammalian target of rapamycin complex I (mTORC1), and extends lifespan in wild-type (WT) C. elegans6-8.
Our lab previously demonstrated that these interventions can act in the same genetic pathway to extend lifespan. Specifically, we have shown that deguelin requires fmo-2 to promote longevity in C. elegans, suggesting that fmo-2 acts downstream of deguelin-mediated lifespan extension6. Furthermore, we established that fmo-2 also requires fmo-4 for its overexpression to extend lifespan4. Given these interconnected relationships, here we investigate whether deguelin and fmo-4 genetically interact in the context of longevity. Interestingly, our recent publication shows that fmo-4 extends lifespan downstream of the inhibition of mTORC1 (rsks-1 RNA interference) and has genetic ties to mitochondrial metabolism4. Considering these similarities between fmo-4 and deguelin, we hypothesized that deguelin may extend lifespan and promote healthspan through the induction of fmo-4, and we sought to investigate the downstream effectors involved in this process.
To address this hypothesis, we first examined the effects of deguelin on WT C. elegans lifespan. Our lab published that deguelin, in addition to multiple other compounds, extends lifespan of WT worms6, and so we aimed to validate these data using a range of concentrations. Our results confirm that deguelin extends WT lifespan in a dose-dependent manner (Figure 1A). To further investigate the quality of life during this extended lifespan, we assessed the healthspan of deguelin-treated worms. Our data show that deguelin not only extends lifespan but also promotes healthspan in middle-aged (day 8 of adulthood) WT worms (Figure 1B), indicating that the compound may improve overall health and functionality during aging.
To explore the molecular mechanisms underlying deguelin's effects, we focused on the potential involvement of fmo-4. We recently found that fmo-4 is required for mTOR pathway gene rsks-1-RNAi-mediated longevity, and that it extends lifespan by regulating calcium signaling between the ER and mitochondria4. This is interesting because deguelin is an inhibitor of both mTORC1 and mitochondrial complex I7, providing more evidence for a potential interaction between the two longevity interventions. Using a transcriptional fmo-4::mCherry reporter, we observed that deguelin treatment induces the expression of fmo-4 in C. elegans by ~2-fold. Fluorescent images and quantification reveal this significant increase in fmo-4 expression in deguelin-treated worms compared to the DMSO-treated control worms (Figure 1C-D). This induction suggests that fmo-4 is regulated by deguelin and may play a crucial role in mediating the longevity-promoting effects of deguelin. To confirm the requirement of fmo-4 in deguelin-mediated lifespan extension, we performed a lifespan assay using fmo-4 knockout (KO) worms. Our results demonstrate that the lifespan-extending effect of deguelin is abolished in fmo-4 KO worms (Figure 1E), indicating that fmo-4 is indeed necessary for and downstream of deguelin to extend lifespan in C. elegans. Similarly, we found that fmo-4 is also required for deguelin to promote healthspan in day 8 adult worms (Figure 1F), further supporting the critical role of fmo-4 in mediating the beneficial effects of deguelin on aging.
To gain insight into the downstream mechanisms of deguelin-mediated longevity, we investigated the involvement of mcu-1, a downstream effector of fmo-4-mediated longevity4,9. mcu-1 is a mitochondrial calcium uniporter that has been implicated in aging, stress response, and calcium regulation4,9. Since fmo-4 is required for the longevity and healthspan effect seen with deguelin treatment, and since mcu-1 acts downstream of fmo-44, we hypothesized that mcu-1 would also be required for the benefits of deguelin treatment. Our data show that mcu-1 is required for deguelin-mediated longevity (Figure 1G), suggesting that the effects of deguelin on lifespan extension may involve modulation of mitochondrial calcium homeostasis through the fmo-4/mcu-1 pathway.
While our findings provide compelling evidence for the role of deguelin in promoting longevity and healthspan in C. elegans through the induction of fmo-4, there are several limitations to consider. First, the exact mechanism by which deguelin induces fmo-4 expression remains to be elucidated. It is possible that deguelin, fmo-2, and fmo-4 are working in the same genetic pathway to promote these health benefits but more work needs to be done to confirm this involvement. Additionally, further research is needed to determine whether the effects of deguelin on lifespan and healthspan are conserved in other organisms, including mammals. Finally, potential off-target effects of deguelin and long-term consequences of its administration should be carefully evaluated in future studies.
In summary, our data demonstrate that deguelin extends lifespan and promotes healthspan in C. elegans in an fmo-4-dependent manner. We find that deguelin induces the expression of fmo-4 and that both fmo-4 and its downstream effector mcu-1 are required for deguelin-mediated longevity. These findings contribute to our understanding of the molecular mechanisms underlying lifespan extension and highlight the potential of deguelin as a pro-longevity compound. Future directions for this research include investigating the upstream regulators of fmo-4 induction by deguelin, exploring the conservation of this pathway in higher organisms, and evaluating the potential of deguelin or related compounds as interventions to promote healthy aging in humans.
","references":[{"reference":"Guo J, Huang X, Dou L, Yan M, Shen T, Tang W, Li J. 2022. Aging and aging-related diseases: from molecular mechanisms to interventions and treatments. Signal Transduct Target Ther 7(1): 391.
","pubmedId":"36522308","doi":""},{"reference":"Kenyon CJ. 2010. The genetics of ageing. Nature 464(7288): 504-12.
","pubmedId":"20336132","doi":""},{"reference":"Leiser SF, Miller H, Rossner R, Fletcher M, Leonard A, Primitivo M, et al., Kaeberlein M. 2015. Cell nonautonomous activation of flavin-containing monooxygenase promotes longevity and health span. Science 350(6266): 1375-1378.
","pubmedId":"26586189","doi":""},{"reference":"Tuckowski AM, Beydoun S, Kitto ES, Bhat A, Howington MB, Sridhar A, et al., Leiser. 2025. fmo-4 promotes longevity and stress resistance via ER to mitochondria calcium regulation in C. elegans. eLife 13: 10.7554/elife.99971.
","pubmedId":"","doi":"10.7554/eLife.99971"},{"reference":"Choi HS, Bhat A, Howington MB, Schaller ML, Cox RL, Huang S, et al., Leiser SF. 2023. FMO rewires metabolism to promote longevity through tryptophan and one carbon metabolism in C. elegans. Nat Commun 14(1): 562.
","pubmedId":"36732543","doi":""},{"reference":"Huang S, Cox RL, Tuckowski A, Beydoun S, Bhat A, Howington MB, et al., Leiser SF. 2024. Fmo induction as a tool to screen for pro-longevity drugs. Geroscience 46(5): 4689-4706.
","pubmedId":"38787463","doi":""},{"reference":"Carpenter EL, Chagani S, Nelson D, Cassidy PB, Laws M, Ganguli-Indra G, Indra AK. 2019. Mitochondrial complex I inhibitor deguelin induces metabolic reprogramming and sensitizes vemurafenib-resistant BRAF(V600E) mutation bearing metastatic melanoma cells. Mol Carcinog 58(9): 1680-1690.
","pubmedId":"31211467","doi":""},{"reference":"Xu H, Li X, Ding W, Zeng X, Kong H, Wang H, Xie W. 2015. Deguelin induces the apoptosis of lung cancer cells through regulating a ROS driven Akt pathway. Cancer Cell Int 15: 25.
","pubmedId":"25741219","doi":""},{"reference":"Marchi S, Pinton P. 2014. The mitochondrial calcium uniporter complex: molecular components, structure and physiopathological implications. J Physiol 592(5): 829-39.
","pubmedId":"24366263","doi":""}],"suggestedReviewer":{"name":"Kristopher Burkewitz kristopher.burkewitz@Vanderbilt.Edu
Mark McCormick mmccormick@salud.unm.edu
Jeremy Van Raamsdonk jeremy.vanraamsdonk@mcgill.ca
","WBId":""},"title":"Deguelin promotes longevity and healthspan through C. elegans fmo-4
","reviews":[]},{"id":"954efe35-3241-4fc5-a8f7-88b2fe5b2d3a","decisionLetter":"\n\n Dear Authors,\n
\n\n Congratulations on your new publication! We are pleased to let you know that your microPublication is \n now available online. You can access it here: https://micropublication.org/journals/biology/micropub-biology-001548\n
\n\n Your article will be sent to PubMed Central in 2 weeks. Please make sure there are no typos, errors or \n omissions in your article, including your title, author names, affiliations, reagents, etc. in addition \n to your reported results. If you want to make corrections, contact us with the title of your article and \n your requested edits at editors@micropublication.org.\n
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\n\n Thank you for submitting your data to us. We look forward to working with you again.\n
\n\n For your records, this is your article's citation:
\n \"Tuckowski AM, Huang S, Chambers K, Buscher B, Leiser SF. 2025. Deguelin promotes longevity and healthspan through C. elegans fmo-4. microPublication Biology. 10.17912/micropub.biology.001548.\"\n
\n Best wishes,\n
\n\n The microPublication Team\n
\n ","decision":"publish","submitted":true,"abstract":"There are multiple approaches to longevity interventions in Caenorhabditis elegans, including genetic factors that are necessary or sufficient for lifespan extension and pharmacological agents that modify physiology to extend lifespan. Many pharmacological interventions act through known genetic pathways to promote longevity. Here, we show that the mitochondrial complex I inhibitor, deguelin, promotes lifespan extension and healthspan in an fmo-4-dependent manner. Our results confirm that deguelin increases lifespan and indicate that deguelin induces and requires multiple FMO enzymes to extend lifespan in C. elegans, suggesting these enzymes may promote longevity in a coordinated fashion.
","acknowledgements":"","authors":[{"affiliations":["University of Michigan"],"credit":["conceptualization","formalAnalysis","investigation","methodology","writing_originalDraft","writing_reviewEditing"],"email":"atuckow@umich.edu","firstName":"Angela M","lastName":"Tuckowski","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9125-4780"},{"affiliations":["Kansas State University"],"credit":["conceptualization","writing_reviewEditing"],"email":"shijiaoh@ksu.edu","firstName":"Shijiao","lastName":"Huang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["methodology","writing_reviewEditing"],"email":"chamberskelly5698@gmail.com","firstName":"Kelly","lastName":"Chambers","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["methodology","writing_reviewEditing"],"email":"bubrando@umich.edu","firstName":"Brandon","lastName":"Buscher","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["writing_reviewEditing","supervision","resources"],"email":"leiser@umich.edu","firstName":"Scott F","lastName":"Leiser","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"comments":"We thank the reviewer for their thoughtful and constructive comments on our manuscript \"Deguelin promotes longevity and healthspan through C. elegans fmo-4.\" We appreciate the time and effort they have devoted to providing this feedback. We have considered the comments and have addressed them in a revised manuscript as detailed below.
Comment 1: In both x-axis labels of Figure 1F I believe that \"fmo-4\" should be italicized.
Response 1: We thank the reviewer for this helpful revision. We have made the change as suggested in the Figure 1F x-axis labels.
Comment 2: \"A gene family of particular interest are the flavin-containing monooxygenase (fmo) genes. From this family fmo-2 and fmo-4 are both sufficient and necessary for lifespan extension in C. elegans3,4.\" Here the footnotes for references 3 and 4 have been inadvertently italicized probably as they follow \"C. elegans.\"
Response 2: Thank you for identifying this. We have made the change as suggested. Additionally, we made sure to look through the rest of the manuscript for similar issues and fixed the italicized footnotes here: \"...inhibits mammalian target of rapamycin complex I (mTORC1), and extends lifespan in wild-type (WT) C. elegans6-8.\"
","dataTable":null,"disclaimer":true,"funding":"This work was supported by grants from NIH. AMT was supported by NIH T32AG000114, NIH 5T32GM007315, and the University of Michigan Rackham Research Grant. SFL was supported by R01AG075061 and the Glenn Foundation for Medical Research.
","image":{"name":"Figure Revised microPub Tuckowski.jpg","url":"https://portal.micropublication.org/uploads/1659665096a38cdd38c009e8c4028853.jpg"},"imageCaption":"(A) Lifespan assessment of wild-type (WT) worms exposed to DMSO control, 1 µM deguelin, 5 µM deguelin, or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis). (B) Healthspan analysis of wild-type (WT) worms exposed to DMSO control or 10 µM deguelin thrashing in a drop of M9 solution for 30 seconds on days 1 and 8 of adulthood (n = ~10 worms per condition, three replicate experiments). (C) Fluorescence intensity of fmo-4p::mCherry transcriptional reporter worms exposed to DMSO control or 10 µM deguelin (n = ~20 worms per condition, three replicate experiments), quantified in (D). (E) Lifespan assessment of wild-type (WT) and fmo-4 knockout (KO) worms exposed to DMSO control or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis and significant interactions between the condition of interest and genotype was determined at p < 0.01 using Cox regression analysis). (F) Healthspan analysis of wild-type (WT) and fmo-4 knockout (KO) worms exposed to DMSO control or 10 µM deguelin thrashing in a drop of M9 solution for 30 seconds on days 1 and 8 of adulthood (n = ~10 worms per condition, three replicate experiments). (G) Lifespan assessment of wild-type (WT) and mcu-1 knockout (KO) worms exposed to DMSO control or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis and significant interactions between condition of interest and genotype was determined at p < 0.01 using Cox regression analysis). For healthspan and imaging experiments, * denotes significant change at p < 0.05 using unpaired two-tailed t test. NS = not significant. All replicate data can be found in the Source Data files.
","imageTitle":"Deguelin requires fmo-4 to promote lifespan extension and improve healthspan in C. elegans.
","laboratory":{"name":"","WBId":""},"methods":"Strains and Maintenance
Standard C. elegans cultivation procedures were used as previously described3. Worm strains were maintained on solid nematode growth medium (NGM) using E. coli OP50 throughout life. Worms were transferred using a platinum wire. All worm strains were maintained at 20°C.
Lifespan Assays
Gravid adult worms were placed on NGM plates seeded with E. coli OP50 for three hours. Then the adults were removed and eggs were allowed to hatch and develop to day 1 adulthood at 20°C. Adult worms were transferred to NGM plates containing 25 µg/mL carbenicillin, floxuridine (FUdR), and either dimethylsulfoxide (DMSO), 1 µM deguelin (Sigma, D0817), 5 µM deguelin, or 10 µM deguelin. Additionally, these plates were seeded with 200 µL of paraformaldehyde (PFA) killed E. coli OP50 at a concentration of 3x. Approximately 70 worms were transferred to fresh plates on day 1, day 2, day 4, and day 6 of adulthood. Two plates per strain per condition were tested per replicate experiment. Experimental animals were scored every 2-3 days and considered dead when they did not move in response to prodding under a dissection microscope. Worms that crawled off the plate were not considered, but ruptured worms were considered as previously described3. Three replicates were performed for each lifespan assay.
Thrashing Assay
Worms were synchronized by placing 10 gravid adult worms on NGM plates seeded with E. coli OP50 and allowing them to lay eggs for 2 hours at 20°C. The gravid adults were removed and the eggs were allowed to hatch and develop at 20°C until larval stage 2 (L2). At this stage, the L2 worms were transferred to NGM plates containing either DMSO or 10 µM deguelin, seeded with paraformaldehyde killed E. coli OP50. On day 1 adulthood, worms were placed in a drop of M9 solution, as previously described. The body bends were counted at maximum rate for 30 seconds. Thrashing was assayed on day 1 and day 8 of adulthood. The worms that were not used for the day 1 assay were transferred to fresh plates containing either DMSO or deguelin two times until they were ready to be assayed. Three replicates were performed. Data were analyzed in GraphPad Prism using unpaired two-tailed t tests with Welch's correction.
fmo-4 Induction on Deguelin
Gravid fmo-4p::mCherry transcriptional reporter adult animals were placed on NGM plates seeded with E. coli OP50. After 3 hours, adults were removed and the eggs were allowed to develop at 20°C until they reached larval stage 4 (L4). Then 30 of the L4 worms were transferred to NGM plates containing either DMSO or 10 µM deguelin and seeded with paraformaldehyde (PFA) killed E. coli OP50. The worms were incubated for 24 hours at 20°C. Then ~20 worms per condition were picked off these plates and added to unseeded NGM plates, anesthetized in 0.5 M sodium azide (Sigma), and imaged at 6.3x magnification with the LASx software and Leica scope using the mCherry fluorescence channel. Three replicates were performed. Each worm was measured for fluorescence in ImageJ. Data were analyzed in GraphPad Prism using t tests.
Statistical Analyses
Log-rank test was used to derive p-value for lifespan assays using p < 0.05 cut-off threshold compared to DMSO or wild-type controls. Cox regression was also used to assess interactions between genotype and condition for lifespans using p < 0.01 cut-off threshold compared to controls. Supplemental Data 1 provide the results of the Log-rank test and Cox regression analyses, which were run in RStudio.
Strain Name | Genotype | Source |
WT or N2 | WT | CGC |
CGC | ||
CGC | ||
fmo-4 transcriptional reporter | fmo-4p::mCherry | Suny Bioscience |
Reagent | Source | Catalog # |
Deguelin | Sigma | D0817-5MG |
DMSO | Fisher Scientific | BP213-100 |
Sodium Azide | Sigma | S2002-5G |
Aging is a complex biological process that affects all living organisms, leading to a decline in physiological functions and an increased susceptibility to diseases1. Studying the aging process is crucial because it allows for a better understanding of how to prevent or delay the onset of multiple age-related chronic diseases simultaneously. The nematode Caenorhabditis elegans (C. elegans) has emerged as a powerful model organism for studying aging due to its short lifespan, well-characterized genetics, and conserved aging pathways2. Recent research has focused on identifying longevity interventions in C. elegans, including genetic factors and pharmacological agents that can extend lifespan and improve healthspan, potentially offering insights into interventions that could promote healthy aging in humans.
A gene family of particular interest are the flavin-containing monooxygenase (fmo) genes. From this family, fmo-2 and fmo-4 are both sufficient and necessary for lifespan extension in C. elegans3,4. These genes play crucial roles in altering cellular metabolism, including one carbon metabolism5 and calcium regulation between the endoplasmic reticulum and mitochondria4, contributing to the longevity phenotype. Pharmacologically, compounds like deguelin, a natural rotenoid isolated from plants of the Leguminosae family, have gained attention for their potential to extend lifespan through the manipulation or inhibition of cellular processes6-8. For instance, deguelin treatment has been successfully tested as an anti-tumor agent in human cells lines, inhibits mitochondrial complex I, inhibits mammalian target of rapamycin complex I (mTORC1), and extends lifespan in wild-type (WT) C. elegans6-8.
Our lab previously demonstrated that these interventions can act in the same genetic pathway to extend lifespan. Specifically, we have shown that deguelin requires fmo-2 to promote longevity in C. elegans, suggesting that fmo-2 acts downstream of deguelin-mediated lifespan extension6. Furthermore, we established that fmo-2 also requires fmo-4 for its overexpression to extend lifespan4. Given these interconnected relationships, here we investigate whether deguelin and fmo-4 genetically interact in the context of longevity. Interestingly, our recent publication shows that fmo-4 extends lifespan downstream of the inhibition of mTORC1 (rsks-1 RNA interference) and has genetic ties to mitochondrial metabolism4. Considering these similarities between fmo-4 and deguelin, we hypothesized that deguelin may extend lifespan and promote healthspan through the induction of fmo-4, and we sought to investigate the downstream effectors involved in this process.
To address this hypothesis, we first examined the effects of deguelin on WT C. elegans lifespan. Our lab published that deguelin, in addition to multiple other compounds, extends lifespan of WT worms6, and so we aimed to validate these data using a range of concentrations. Our results confirm that deguelin extends WT lifespan in a dose-dependent manner (Figure 1A). To further investigate the quality of life during this extended lifespan, we assessed the healthspan of deguelin-treated worms. Our data show that deguelin not only extends lifespan but also promotes healthspan in middle-aged (day 8 of adulthood) WT worms (Figure 1B), indicating that the compound may improve overall health and functionality during aging.
To explore the molecular mechanisms underlying deguelin's effects, we focused on the potential involvement of fmo-4. We recently found that fmo-4 is required for mTOR pathway gene rsks-1-RNAi-mediated longevity, and that it extends lifespan by regulating calcium signaling between the ER and mitochondria4. This is interesting because deguelin is an inhibitor of both mTORC1 and mitochondrial complex I7, providing more evidence for a potential interaction between the two longevity interventions. Using a transcriptional fmo-4::mCherry reporter, we observed that deguelin treatment induces the expression of fmo-4 in C. elegans by ~2-fold. Fluorescent images and quantification reveal this significant increase in fmo-4 expression in deguelin-treated worms compared to the DMSO-treated control worms (Figure 1C-D). This induction suggests that fmo-4 is regulated by deguelin and may play a crucial role in mediating the longevity-promoting effects of deguelin. To confirm the requirement of fmo-4 in deguelin-mediated lifespan extension, we performed a lifespan assay using fmo-4 knockout (KO) worms. Our results demonstrate that the lifespan-extending effect of deguelin is abolished in fmo-4 KO worms (Figure 1E), indicating that fmo-4 is indeed necessary for and downstream of deguelin to extend lifespan in C. elegans. Similarly, we found that fmo-4 is also required for deguelin to promote healthspan in day 8 adult worms (Figure 1F), further supporting the critical role of fmo-4 in mediating the beneficial effects of deguelin on aging.
To gain insight into the downstream mechanisms of deguelin-mediated longevity, we investigated the involvement of mcu-1, a downstream effector of fmo-4-mediated longevity4,9. mcu-1 is a mitochondrial calcium uniporter that has been implicated in aging, stress response, and calcium regulation4,9. Since fmo-4 is required for the longevity and healthspan effect seen with deguelin treatment, and since mcu-1 acts downstream of fmo-44, we hypothesized that mcu-1 would also be required for the benefits of deguelin treatment. Our data show that mcu-1 is required for deguelin-mediated longevity (Figure 1G), suggesting that the effects of deguelin on lifespan extension may involve modulation of mitochondrial calcium homeostasis through the fmo-4/mcu-1 pathway.
While our findings provide compelling evidence for the role of deguelin in promoting longevity and healthspan in C. elegans through the induction of fmo-4, there are several limitations to consider. First, the exact mechanism by which deguelin induces fmo-4 expression remains to be elucidated. It is possible that deguelin, fmo-2, and fmo-4 are working in the same genetic pathway to promote these health benefits but more work needs to be done to confirm this involvement. Additionally, further research is needed to determine whether the effects of deguelin on lifespan and healthspan are conserved in other organisms, including mammals. Finally, potential off-target effects of deguelin and long-term consequences of its administration should be carefully evaluated in future studies.
In summary, our data demonstrate that deguelin extends lifespan and promotes healthspan in C. elegans in an fmo-4-dependent manner. We find that deguelin induces the expression of fmo-4 and that both fmo-4 and its downstream effector mcu-1 are required for deguelin-mediated longevity. These findings contribute to our understanding of the molecular mechanisms underlying lifespan extension and highlight the potential of deguelin as a pro-longevity compound. Future directions for this research include investigating the upstream regulators of fmo-4 induction by deguelin, exploring the conservation of this pathway in higher organisms, and evaluating the potential of deguelin or related compounds as interventions to promote healthy aging in humans.
","references":[{"reference":"Guo J, Huang X, Dou L, Yan M, Shen T, Tang W, Li J. 2022. Aging and aging-related diseases: from molecular mechanisms to interventions and treatments. Signal Transduct Target Ther 7(1): 391.
","pubmedId":"36522308","doi":""},{"reference":"Kenyon CJ. 2010. The genetics of ageing. Nature 464(7288): 504-12.
","pubmedId":"20336132","doi":""},{"reference":"Leiser SF, Miller H, Rossner R, Fletcher M, Leonard A, Primitivo M, et al., Kaeberlein M. 2015. Cell nonautonomous activation of flavin-containing monooxygenase promotes longevity and health span. Science 350(6266): 1375-1378.
","pubmedId":"26586189","doi":""},{"reference":"Tuckowski AM, Beydoun S, Kitto ES, Bhat A, Howington MB, Sridhar A, et al., Leiser. 2025. fmo-4 promotes longevity and stress resistance via ER to mitochondria calcium regulation in C. elegans. eLife 13: 10.7554/elife.99971.
","pubmedId":"","doi":"10.7554/eLife.99971"},{"reference":"Choi HS, Bhat A, Howington MB, Schaller ML, Cox RL, Huang S, et al., Leiser SF. 2023. FMO rewires metabolism to promote longevity through tryptophan and one carbon metabolism in C. elegans. Nat Commun 14(1): 562.
","pubmedId":"36732543","doi":""},{"reference":"Huang S, Cox RL, Tuckowski A, Beydoun S, Bhat A, Howington MB, et al., Leiser SF. 2024. Fmo induction as a tool to screen for pro-longevity drugs. Geroscience 46(5): 4689-4706.
","pubmedId":"38787463","doi":""},{"reference":"Carpenter EL, Chagani S, Nelson D, Cassidy PB, Laws M, Ganguli-Indra G, Indra AK. 2019. Mitochondrial complex I inhibitor deguelin induces metabolic reprogramming and sensitizes vemurafenib-resistant BRAF(V600E) mutation bearing metastatic melanoma cells. Mol Carcinog 58(9): 1680-1690.
","pubmedId":"31211467","doi":""},{"reference":"Xu H, Li X, Ding W, Zeng X, Kong H, Wang H, Xie W. 2015. Deguelin induces the apoptosis of lung cancer cells through regulating a ROS driven Akt pathway. Cancer Cell Int 15: 25.
","pubmedId":"25741219","doi":""},{"reference":"Marchi S, Pinton P. 2014. The mitochondrial calcium uniporter complex: molecular components, structure and physiopathological implications. J Physiol 592(5): 829-39.
","pubmedId":"24366263","doi":""}],"suggestedReviewer":{"name":"Kristopher Burkewitz kristopher.burkewitz@Vanderbilt.Edu
Mark McCormick mmccormick@salud.unm.edu
Jeremy Van Raamsdonk jeremy.vanraamsdonk@mcgill.ca
","WBId":""},"title":"Deguelin promotes longevity and healthspan through C. elegans fmo-4
","reviews":[]},{"id":"32538c53-d04d-48f5-8a1a-731af10d058a","decisionLetter":"\n\n Dear Authors,\n
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\n \"Tuckowski AM, Huang S, Chambers K, Buscher B, Leiser SF. 2025. Deguelin promotes longevity and healthspan through C. elegans fmo-4. microPublication Biology. 10.17912/micropub.biology.001548.\"\n
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\n ","decision":"publish","submitted":true,"abstract":"There are multiple approaches to longevity interventions in Caenorhabditis elegans, including genetic factors that are necessary or sufficient for lifespan extension and pharmacological agents that modify physiology to extend lifespan. Many pharmacological interventions act through known genetic pathways to promote longevity. Here, we show that the mitochondrial complex I inhibitor, deguelin, promotes lifespan extension and healthspan in an fmo-4-dependent manner. Our results confirm that deguelin increases lifespan and indicate that deguelin induces and requires multiple FMO enzymes to extend lifespan in C. elegans, suggesting these enzymes may promote longevity in a coordinated fashion.
","acknowledgements":"","authors":[{"affiliations":["University of Michigan"],"credit":["conceptualization","formalAnalysis","investigation","methodology","writing_originalDraft","writing_reviewEditing"],"email":"atuckow@umich.edu","firstName":"Angela M","lastName":"Tuckowski","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9125-4780"},{"affiliations":["Kansas State University"],"credit":["conceptualization","writing_reviewEditing"],"email":"shijiaoh@ksu.edu","firstName":"Shijiao","lastName":"Huang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["methodology","writing_reviewEditing"],"email":"chamberskelly5698@gmail.com","firstName":"Kelly","lastName":"Chambers","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["methodology","writing_reviewEditing"],"email":"bubrando@umich.edu","firstName":"Brandon","lastName":"Buscher","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Michigan"],"credit":["writing_reviewEditing","supervision","resources"],"email":"leiser@umich.edu","firstName":"Scott F","lastName":"Leiser","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"comments":"We thank the reviewer for their thoughtful and constructive comments on our manuscript \"Deguelin promotes longevity and healthspan through C. elegans fmo-4.\" We appreciate the time and effort they have devoted to providing this feedback. We have considered the comments and have addressed them in a revised manuscript as detailed below.
Comment 1: In both x-axis labels of Figure 1F I believe that \"fmo-4\" should be italicized.
Response 1: We thank the reviewer for this helpful revision. We have made the change as suggested in the Figure 1F x-axis labels.
Comment 2: \"A gene family of particular interest are the flavin-containing monooxygenase (fmo) genes. From this family fmo-2 and fmo-4 are both sufficient and necessary for lifespan extension in C. elegans3,4.\" Here the footnotes for references 3 and 4 have been inadvertently italicized probably as they follow \"C. elegans.\"
Response 2: Thank you for identifying this. We have made the change as suggested. Additionally, we made sure to look through the rest of the manuscript for similar issues and fixed the italicized footnotes here: \"...inhibits mammalian target of rapamycin complex I (mTORC1), and extends lifespan in wild-type (WT) C. elegans6-8.\"
","dataTable":{"name":null,"url":null},"disclaimer":true,"funding":"This work was supported by grants from NIH. AMT was supported by NIH T32AG000114, NIH 5T32GM007315, and the University of Michigan Rackham Research Grant. SFL was supported by R01AG075061 and the Glenn Foundation for Medical Research.
","image":{"name":"Tuckowski Leiser Micropublication Edit Figure 1E.jpg","url":"https://portal.micropublication.org/uploads/e7504a47514ab054f7b719f01f4c8d06.jpg"},"imageCaption":"(A) Lifespan assessment of wild-type (WT) worms exposed to DMSO control, 1 µM deguelin, 5 µM deguelin, or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis). (B) Healthspan analysis of wild-type (WT) worms exposed to DMSO control or 10 µM deguelin thrashing in a drop of M9 solution for 30 seconds on days 1 and 8 of adulthood (n = ~10 worms per condition, three replicate experiments). (C) Fluorescence intensity of fmo-4p::mCherry transcriptional reporter worms exposed to DMSO control or 10 µM deguelin (n = ~20 worms per condition, three replicate experiments), quantified in (D). (E) Lifespan assessment of wild-type (WT) and fmo-4 knockout (KO) worms exposed to DMSO control or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis and significant interactions between the condition of interest and genotype was determined at p < 0.01 using Cox regression analysis). (F) Healthspan analysis of wild-type (WT) and fmo-4 knockout (KO) worms exposed to DMSO control or 10 µM deguelin thrashing in a drop of M9 solution for 30 seconds on days 1 and 8 of adulthood (n = ~10 worms per condition, three replicate experiments). (G) Lifespan assessment of wild-type (WT) and mcu-1 knockout (KO) worms exposed to DMSO control or 10 µM deguelin (n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p < 0.05 using log-rank analysis and significant interactions between condition of interest and genotype was determined at p < 0.01 using Cox regression analysis). For healthspan and imaging experiments, * denotes significant change at p < 0.05 using unpaired two-tailed t test. NS = not significant. All replicate data can be found in the Source Data files.
","imageTitle":"Deguelin requires fmo-4 to promote lifespan extension and improve healthspan in C. elegans.
","laboratory":{"name":"","WBId":""},"methods":"Strains and Maintenance
Standard C. elegans cultivation procedures were used as previously described3. Worm strains were maintained on solid nematode growth medium (NGM) using E. coli OP50 throughout life. Worms were transferred using a platinum wire. All worm strains were maintained at 20°C.
Lifespan Assays
Gravid adult worms were placed on NGM plates seeded with E. coli OP50 for three hours. Then the adults were removed and eggs were allowed to hatch and develop to day 1 adulthood at 20°C. Adult worms were transferred to NGM plates containing 25 µg/mL carbenicillin, floxuridine (FUdR), and either dimethylsulfoxide (DMSO), 1 µM deguelin (Sigma, D0817), 5 µM deguelin, or 10 µM deguelin. Additionally, these plates were seeded with 200 µL of paraformaldehyde (PFA) killed E. coli OP50 at a concentration of 3x. Approximately 70 worms were transferred to fresh plates on day 1, day 2, day 4, and day 6 of adulthood. Two plates per strain per condition were tested per replicate experiment. Experimental animals were scored every 2-3 days and considered dead when they did not move in response to prodding under a dissection microscope. Worms that crawled off the plate were not considered, but ruptured worms were considered as previously described3. Three replicates were performed for each lifespan assay.
Thrashing Assay
Worms were synchronized by placing 10 gravid adult worms on NGM plates seeded with E. coli OP50 and allowing them to lay eggs for 2 hours at 20°C. The gravid adults were removed and the eggs were allowed to hatch and develop at 20°C until larval stage 2 (L2). At this stage, the L2 worms were transferred to NGM plates containing either DMSO or 10 µM deguelin, seeded with paraformaldehyde killed E. coli OP50. On day 1 adulthood, worms were placed in a drop of M9 solution, as previously described. The body bends were counted at maximum rate for 30 seconds. Thrashing was assayed on day 1 and day 8 of adulthood. The worms that were not used for the day 1 assay were transferred to fresh plates containing either DMSO or deguelin two times until they were ready to be assayed. Three replicates were performed. Data were analyzed in GraphPad Prism using unpaired two-tailed t tests with Welch's correction.
fmo-4 Induction on Deguelin
Gravid fmo-4p::mCherry transcriptional reporter adult animals were placed on NGM plates seeded with E. coli OP50. After 3 hours, adults were removed and the eggs were allowed to develop at 20°C until they reached larval stage 4 (L4). Then 30 of the L4 worms were transferred to NGM plates containing either DMSO or 10 µM deguelin and seeded with paraformaldehyde (PFA) killed E. coli OP50. The worms were incubated for 24 hours at 20°C. Then ~20 worms per condition were picked off these plates and added to unseeded NGM plates, anesthetized in 0.5 M sodium azide (Sigma), and imaged at 6.3x magnification with the LASx software and Leica scope using the mCherry fluorescence channel. Three replicates were performed. Each worm was measured for fluorescence in ImageJ. Data were analyzed in GraphPad Prism using t tests.
Statistical Analyses
Log-rank test was used to derive p-value for lifespan assays using p < 0.05 cut-off threshold compared to DMSO or wild-type controls. Cox regression was also used to assess interactions between genotype and condition for lifespans using p < 0.01 cut-off threshold compared to controls. Supplemental Data 1 provide the results of the Log-rank test and Cox regression analyses, which were run in RStudio.
Strain Name | Genotype | Source |
WT or N2 | WT | CGC |
CGC | ||
CGC | ||
fmo-4 transcriptional reporter | fmo-4p::mCherry | Suny Bioscience |
Reagent | Source | Catalog # |
Deguelin | Sigma | D0817-5MG |
DMSO | Fisher Scientific | BP213-100 |
Sodium Azide | Sigma | S2002-5G |
Aging is a complex biological process that affects all living organisms, leading to a decline in physiological functions and an increased susceptibility to diseases1. Studying the aging process is crucial because it allows for a better understanding of how to prevent or delay the onset of multiple age-related chronic diseases simultaneously. The nematode Caenorhabditis elegans (C. elegans) has emerged as a powerful model organism for studying aging due to its short lifespan, well-characterized genetics, and conserved aging pathways2. Recent research has focused on identifying longevity interventions in C. elegans, including genetic factors and pharmacological agents that can extend lifespan and improve healthspan, potentially offering insights into interventions that could promote healthy aging in humans.
A gene family of particular interest are the flavin-containing monooxygenase (fmo) genes. From this family, fmo-2 and fmo-4 are both sufficient and necessary for lifespan extension in C. elegans3,4. These genes play crucial roles in altering cellular metabolism, including one carbon metabolism5 and calcium regulation between the endoplasmic reticulum and mitochondria4, contributing to the longevity phenotype. Pharmacologically, compounds like deguelin, a natural rotenoid isolated from plants of the Leguminosae family, have gained attention for their potential to extend lifespan through the manipulation or inhibition of cellular processes6-8. For instance, deguelin treatment has been successfully tested as an anti-tumor agent in human cells lines, inhibits mitochondrial complex I, inhibits mammalian target of rapamycin complex I (mTORC1), and extends lifespan in wild-type (WT) C. elegans6-8.
Our lab previously demonstrated that these interventions can act in the same genetic pathway to extend lifespan. Specifically, we have shown that deguelin requires fmo-2 to promote longevity in C. elegans, suggesting that fmo-2 acts downstream of deguelin-mediated lifespan extension6. Furthermore, we established that fmo-2 also requires fmo-4 for its overexpression to extend lifespan4. Given these interconnected relationships, here we investigate whether deguelin and fmo-4 genetically interact in the context of longevity. Interestingly, our recent publication shows that fmo-4 extends lifespan downstream of the inhibition of mTORC1 (rsks-1 RNA interference) and has genetic ties to mitochondrial metabolism4. Considering these similarities between fmo-4 and deguelin, we hypothesized that deguelin may extend lifespan and promote healthspan through the induction of fmo-4, and we sought to investigate the downstream effectors involved in this process.
To address this hypothesis, we first examined the effects of deguelin on WT C. elegans lifespan. Our lab published that deguelin, in addition to multiple other compounds, extends lifespan of WT worms6, and so we aimed to validate these data using a range of concentrations. Our results confirm that deguelin extends WT lifespan in a dose-dependent manner (Figure 1A). To further investigate the quality of life during this extended lifespan, we assessed the healthspan of deguelin-treated worms. Our data show that deguelin not only extends lifespan but also promotes healthspan in middle-aged (day 8 of adulthood) WT worms (Figure 1B), indicating that the compound may improve overall health and functionality during aging.
To explore the molecular mechanisms underlying deguelin's effects, we focused on the potential involvement of fmo-4. We recently found that fmo-4 is required for mTOR pathway gene rsks-1-RNAi-mediated longevity, and that it extends lifespan by regulating calcium signaling between the ER and mitochondria4. This is interesting because deguelin is an inhibitor of both mTORC1 and mitochondrial complex I7, providing more evidence for a potential interaction between the two longevity interventions. Using a transcriptional fmo-4::mCherry reporter, we observed that deguelin treatment induces the expression of fmo-4 in C. elegans by ~2-fold. Fluorescent images and quantification reveal this significant increase in fmo-4 expression in deguelin-treated worms compared to the DMSO-treated control worms (Figure 1C-D). This induction suggests that fmo-4 is regulated by deguelin and may play a crucial role in mediating the longevity-promoting effects of deguelin. To confirm the requirement of fmo-4 in deguelin-mediated lifespan extension, we performed a lifespan assay using fmo-4 knockout (KO) worms. Our results demonstrate that the lifespan-extending effect of deguelin is abolished in fmo-4 KO worms (Figure 1E), indicating that fmo-4 is indeed necessary for and downstream of deguelin to extend lifespan in C. elegans. Similarly, we found that fmo-4 is also required for deguelin to promote healthspan in day 8 adult worms (Figure 1F), further supporting the critical role of fmo-4 in mediating the beneficial effects of deguelin on aging.
To gain insight into the downstream mechanisms of deguelin-mediated longevity, we investigated the involvement of mcu-1, a downstream effector of fmo-4-mediated longevity4,9. mcu-1 is a mitochondrial calcium uniporter that has been implicated in aging, stress response, and calcium regulation4,9. Since fmo-4 is required for the longevity and healthspan effect seen with deguelin treatment, and since mcu-1 acts downstream of fmo-44, we hypothesized that mcu-1 would also be required for the benefits of deguelin treatment. Our data show that mcu-1 is required for deguelin-mediated longevity (Figure 1G), suggesting that the effects of deguelin on lifespan extension may involve modulation of mitochondrial calcium homeostasis through the fmo-4/mcu-1 pathway.
While our findings provide compelling evidence for the role of deguelin in promoting longevity and healthspan in C. elegans through the induction of fmo-4, there are several limitations to consider. First, the exact mechanism by which deguelin induces fmo-4 expression remains to be elucidated. It is possible that deguelin, fmo-2, and fmo-4 are working in the same genetic pathway to promote these health benefits but more work needs to be done to confirm this involvement. Additionally, further research is needed to determine whether the effects of deguelin on lifespan and healthspan are conserved in other organisms, including mammals. Finally, potential off-target effects of deguelin and long-term consequences of its administration should be carefully evaluated in future studies.
In summary, our data demonstrate that deguelin extends lifespan and promotes healthspan in C. elegans in an fmo-4-dependent manner. We find that deguelin induces the expression of fmo-4 and that both fmo-4 and its downstream effector mcu-1 are required for deguelin-mediated longevity. These findings contribute to our understanding of the molecular mechanisms underlying lifespan extension and highlight the potential of deguelin as a pro-longevity compound. Future directions for this research include investigating the upstream regulators of fmo-4 induction by deguelin, exploring the conservation of this pathway in higher organisms, and evaluating the potential of deguelin or related compounds as interventions to promote healthy aging in humans.
","references":[{"reference":"Guo J, Huang X, Dou L, Yan M, Shen T, Tang W, Li J. 2022. Aging and aging-related diseases: from molecular mechanisms to interventions and treatments. Signal Transduct Target Ther 7(1): 391.
","pubmedId":"36522308","doi":""},{"reference":"Kenyon CJ. 2010. The genetics of ageing. Nature 464(7288): 504-12.
","pubmedId":"20336132","doi":""},{"reference":"Leiser SF, Miller H, Rossner R, Fletcher M, Leonard A, Primitivo M, et al., Kaeberlein M. 2015. Cell nonautonomous activation of flavin-containing monooxygenase promotes longevity and health span. Science 350(6266): 1375-1378.
","pubmedId":"26586189","doi":""},{"reference":"Tuckowski AM, Beydoun S, Kitto ES, Bhat A, Howington MB, Sridhar A, et al., Leiser. 2025. fmo-4 promotes longevity and stress resistance via ER to mitochondria calcium regulation in C. elegans. eLife 13: 10.7554/elife.99971.
","pubmedId":"","doi":"10.7554/eLife.99971"},{"reference":"Choi HS, Bhat A, Howington MB, Schaller ML, Cox RL, Huang S, et al., Leiser SF. 2023. FMO rewires metabolism to promote longevity through tryptophan and one carbon metabolism in C. elegans. Nat Commun 14(1): 562.
","pubmedId":"36732543","doi":""},{"reference":"Huang S, Cox RL, Tuckowski A, Beydoun S, Bhat A, Howington MB, et al., Leiser SF. 2024. Fmo induction as a tool to screen for pro-longevity drugs. Geroscience 46(5): 4689-4706.
","pubmedId":"38787463","doi":""},{"reference":"Carpenter EL, Chagani S, Nelson D, Cassidy PB, Laws M, Ganguli-Indra G, Indra AK. 2019. Mitochondrial complex I inhibitor deguelin induces metabolic reprogramming and sensitizes vemurafenib-resistant BRAF(V600E) mutation bearing metastatic melanoma cells. Mol Carcinog 58(9): 1680-1690.
","pubmedId":"31211467","doi":""},{"reference":"Xu H, Li X, Ding W, Zeng X, Kong H, Wang H, Xie W. 2015. Deguelin induces the apoptosis of lung cancer cells through regulating a ROS driven Akt pathway. Cancer Cell Int 15: 25.
","pubmedId":"25741219","doi":""},{"reference":"Marchi S, Pinton P. 2014. The mitochondrial calcium uniporter complex: molecular components, structure and physiopathological implications. J Physiol 592(5): 829-39.
","pubmedId":"24366263","doi":""}],"suggestedReviewer":{"name":"Kristopher Burkewitz kristopher.burkewitz@Vanderbilt.Edu
Mark McCormick mmccormick@salud.unm.edu
Jeremy Van Raamsdonk jeremy.vanraamsdonk@mcgill.ca
","WBId":""},"title":"Deguelin promotes longevity and healthspan through C. elegans fmo-4
","reviews":[]}]}}},"pageContext":{"id":"b49f7c63-0d13-4860-85c2-6c388e18f025","correctionId":"micropub-biology-002391"}}, "staticQueryHashes": ["2114697108"]}