Calorie restriction (CR) is a dietary intervention used to promote health and longevity1,2. CR causes various metabolic changes in both the production and the circulation of metabolites1; however, it remains unclear which altered metabolites account for the physiological benefits of CR. Here we use metabolomics to analyse metabolites that exhibit changes in abundance during CR and perform subsequent functional validation. We show that lithocholic acid (LCA) is one of the metabolites that alone can recapitulate the effects of CR in mice. These effects include activation of AMP-activated protein kinase (AMPK), enhancement of muscle regeneration and rejuvenation of grip strength and running capacity. LCA also activates AMPK and induces life-extending and health-extending effects in Caenorhabditis elegans and Drosophila melanogaster. As C. elegans and D. melanogaster are not able to synthesize LCA, these results indicate that these animals are able to transmit the signalling effects of LCA once administered. Knockout of AMPK abrogates LCA-induced phenotypes in all the three animal models. Together, we identify that administration of the CR-mediated upregulated metabolite LCA alone can confer anti-ageing benefits to metazoans in an AMPK-dependent manner.

Lithocholic acid (LCA) is accumulated in mammals during calorie restriction and it can activate AMP-activated protein kinase (AMPK) to slow down ageing1. However, the molecular details of how LCA activates AMPK and induces these biological effects are unclear. Here we show that LCA enhances the activity of sirtuins to deacetylate and subsequently inhibit vacuolar H+-ATPase (v-ATPase), which leads to AMPK activation through the lysosomal glucose-sensing pathway. Proteomics analyses of proteins that co-immunoprecipitated with sirtuin 1 (SIRT1) identified TUB-like protein 3 (TULP3), a sirtuin-interacting protein2, as a LCA receptor. In detail, LCA-bound TULP3 allosterically activates sirtuins, which then deacetylate the V1E1 subunit of v-ATPase on residues K52, K99 and K191. Muscle-specific expression of a V1E1 mutant (3KR), which mimics the deacetylated state, strongly activates AMPK and rejuvenates muscles in aged mice. In nematodes and flies, LCA depends on the TULP3 homologues tub-1 and ktub, respectively, to activate AMPK and extend lifespan and healthspan. Our study demonstrates that activation of the TULP3-sirtuin-v-ATPase-AMPK pathway by LCA reproduces the benefits of calorie restriction.

Lithocholic acid (LCA), accumulated in the body during calorie restriction (CR), can confer administered metazoans with the ability to activate AMP-activated protein kinase (AMPK) and retard ageing. However, how LCA is signalled to activate AMPK and elicit the biological effects is unclear. Here, we show that LCA can enhance sirtuins (SIRTs) to deacetylate and subsequently inhibit vacuolar H+-ATPase (v-ATPase), thereby triggering AMPK activation via the lysosomal glucose-sensing pathway. Through proteomic analysis of SIRT1-coimmunoprecipitated proteins, we identify and validate that TUB like protein 3 (TULP3) is a constitutive component of SIRTs. Surprisingly, we found that TULP3 is an LCA receptor, and that the LCA-bound TULP3 activates SIRTs. The activated SIRTs in turn deacetylate the V1E1 subunit of v-ATPase on K52, K99 and K191 residues. Muscle-specific expression of the 3KR mutant of V1E1, mimicking the deacetylated state, dominantly activates AMPK and rejuvenates muscles in aged mice. Moreover, LCA once administered also activates AMPK and extends lifespan and healthspan in nematodes and flies, depending on the TULP3 homologues tub-1 and ktub , respectively. Our study thus elucidates that LCA triggers the TULP3-sirtuin-v-ATPase- AMPK route to manifest benefits of calorie restriction.### Competing Interest StatementThe authors have declared no competing interest.

Calorie restriction (CR) is a dietary intervention to promote health and longevity. CR causes various metabolic changes in both the production and circulation of metabolites; however, it remains unclear which of the changed metabolite(s) can account for the physiological benefits of CR. Through metabolomic analysis of metabolites undergoing abundance changes during CR and subsequent functional validation, we found that lithocholic acid (LCA) is the only metabolite that alone can recapitulate the effects of CR, including activation of AMPK and the rejuvenating effects of muscle regeneration, grip strength and running capacity in mice. Interestingly, LCA also activates AMPK and exerts life- and health-extending effects in Caenorhabditis elegans and Drosophila melanogaster , indicating that these animal models are able to transmit the signalling of LCA once administered. Knockout of AMPK abrogates LCA-induced phenotypes, in nematodes and flies, as well as in mice. Together, we have identified that administration of the CR-upregulated metabolite LCA alone can confer anti-ageing benefits to metazoans, in an AMPK-dependent manner.