Senolytics enhance longevity in Caenorhabditis elegans by altering betaine metabolism

  1. Wenning Lan
  2. Xiaolian Xiao
  3. Xiaojing Zhang
  4. Jingjing Nian
  5. Ziran Wang
  6. Yajiao Wu
  7. Dongcheng Zhang
  8. Junkun Chen
  9. Wenqiang Bao
  10. Chutao Li
  11. An Zhu
  12. Yun Zhang
  13. Fangrong Zhang

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Abstract

Aging triggers physiological changes in organisms, which are tightly interlinked to metabolic changes. Senolytics are being developed. However, metabolic responses to natural senescence and the molecular intricacies of how senolytics confer antiaging benefits remain enigmatic. We performed a metabolomics study on natural senescence based on the C.elegans model. The results suggest that age-dependent metabolic changes of natural aging occur in C. elegans . Betaine was identified as a crucial metabolite in the natural aging process. To explore the common pathway coregulated by different senolytics prolonging nematodes’ lifespan, we fed nematodes three antiaging drugs metformin, quercetin, and minocycline. Our data show that the coregulated metabolic pathways associated with aging include the forkhead box transcription factor (FoxO), p38-mitogen-activated protein kinase (MAPK) and the target of rapamycin (mTOR) signaling pathway, etc. Three antiaging drugs raised betaine levels, consistent with high betaine levels in the younger nematode. Supplement of betaine prolonged the lifespan of nematodes via stimulating autophagy and improving antioxidant capacity. Altogether, our data support proof-of-concept evidence that betaine at appropriate concentrations can extend the lifespan of nematodes.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/12660085.

    This manuscript investigates key metabolites in regulating aging and longevity. The authors found that the concentration of betaine in the serum of elderly humans and aged worms is lower than in their younger counterparts, as determined by NMR metabolomics. They also have examined several senolytics including metformin, quercetin, and minocycline, and found that the treated worms had higher betaine level and longer lifespan. Betaine supplementation with certain concentration alone was sufficient to prolong lifespan of worms via stimulating autophagy and improving antioxidant capacity. Overall, the textual content is well written, and the figures are clear. The results support the conclusions. However, there are several concerns that require further attention.

    Major point:

    1. How was the optimal concentration of senolytics decided? The manuscript didn't include thorough analysis using different concentrations of senolytics. It is unclear whether the ones reported in the manuscript are most effective.

    2. There are also changes in choline and glycine, which are important in betaine biosynthesis, in aged worms compared to young worms. What are the levels of choline and glycine in senolytics treated worms?

    3. The current experiments were conducted using liquid culture, which is not the common procedure in longitudinal assays? If growing worms on plates, would senolytics and betaine treatments lead to the same lifespan extension?

    4. The authors need to provide corresponding images of autophagosomes for young worms at day 1 and day 4 as comparison with aged worms.

    5. Details about quantification of metabolites using NMR are missing, such as internal standard and normalization methods. So, please provide information on this quantification.

    Minor point:

    1) There is no explanation of handling progeneis in the method section, so, please provide information on how to manage the progenies in the lifespan assay and on how to measure ROS.

    2) The authors mentioned the utilization of oviposition inhibitors. They should specify the name of the inhibitors, their concentration, and the timing of application.

    3) Figure 2D needs more discussion, such as why micorcylcine group has lower percentage of fast body bends.

    5) It would be better to indicate significance in images showing the lifespan results.

    6) The label of the transmembrane protein is missing in Figure 5. The Figure 5 is also misleading like the readers may think betaine can be derived from these senolytics on the step of senolytics to betaine, please revise it.

    8) There is no access to all the supplementary information, so we are unable to evaluate the related observations or conclusions.

    Competing interests

    The authors declare that they have no competing interests.

  2. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/12661736.

    In this manuscript, Lan et al. investigate metabolomic changes associated with the aging process and explore the impact of senolytic drugs on Caenorhabditis elegans longevity. The authors highlight betaine as a key metabolite regulated by anti-aging drugs including minocycline, metformin, and quercetin. They observed that betaine levels were upregulated in both senolytics-treated worms and young worms. Moreover, they demonstrated that the beneficial effect of betaine in promoting longevity relies on stimulating autophagy and improving antioxidant capacity. In summary, this study suggests that supplementation of betaine at appropriate concentrations can extend the lifespan of Caenorhabditis elegans, providing a useful reference for future translational studies in clinical trials.

    Major points:

    1. Choline levels, an essential substrate for betaine synthesis, are higher at D10 compared to D4, and significantly higher at D1 compared to older worms including D4 and D10. Additionally, choline level shows an opposite trend to betaine in aged worms. The authors should comment on the role of choline levels in the betaine-mediated longevity-promoting effect. Were the enzymes responsible for catalyzing the conversion from choline to betaine aldehyde, and subsequently to betaine, upregulated in aged worms?

    2. The authors should provide the rationale behind the concentrations used for the senolytics. Were these concentrations chosen based on prior knowledge or dose-response experiments?

    3. The minocycline treatment group exhibits a reduced bending rate but a significantly longer lifespan. Could the author explain this observation – differential regulation in health span vs lifespan?

    4. To determine if the AMPK pathway/ROS pathway is essential for the betaine-mediated pro-longevity effect, the authors should knock down or knock out aak-1/2 or reduce SOD antioxidant enzyme activity to test if these manipulations suppress the longevity effect induced by betaine.

    5. The authors need to provide corresponding images of autophagosomes in young worms at D1 and D4.

    6. The authors should provide more details about the quantification of metabolites using NMR, such as internal standards and normalization methods.

    Minor:

    1. There appears to be an alteration in lipofuscin levels (although not significant) on day 4, particularly with Quercetin (images in Figure 2A also indicate the background signal varies between groups) and a much higher variation in D10 DMSO groups. Could the intestinal uptake of the drug itself increase autofluorescent lipofuscin in young worms? It would be helpful if the authors could address or comment on the cause of varied background signals in figure 2A, and high variation in D10 DMSO-treated group.

    2. It would be very helpful to arrange the order of the figures in accordance with the flow of the paper. 3. Betaine exists in both cytosol and mitochondria. It would be helpful if the authors could discuss where betaine acts to exert this pro-longevity effects and illustrate transporters that mediate its transport across the plasma membrane and mitochondria membrane in figure 5.

    4.. It would be better to indicate the significance of the lifespan result figures (figure 2C and 3C).

    5. We did not comment on the content about supplementary figures due to their absence in the biorxiv version.

    Competing interests

    The authors declare that they have no competing interests.

  3. Version published to 10.1101/2023.12.19.572398v1 on bioRxiv