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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.