Lithocholic Acid Activates TULP3-Sirtuin-v-ATPase-AMPK Axis to Enhance Longevity

Abstract

A recent study published in Nature by Lin et al. [1, 2], conducted at Xiamen University identified that lithocholic acid (LCA), a bile acid metabolite, mimics the anti-aging effects of caloric restriction (CR) by activating the TULP3-sirtuin-v-ATPase-AMPK axis. LCA binds to the receptor TUB-like protein 3 (TULP3), triggering allosteric activation of sirtuins (SIRT1-7), which deacetylate lysine residues (K52, K99, K191) on the V1E1 subunit of lysosomal v-ATPase. This deacetylation inhibits v-ATPase activity, activating AMPK through the lysosomal glucose-sensing pathway, fostering muscle rejuvenation in aged mice.

CR has long been associated with enhanced health and longevity in various species including yeasts, worms, flies, and mammals [3]. Although the precise underlying mechanisms of these benefits remain unclear, they potentially involve alterations in multiple metabolic, hormonal, and cellular signaling pathways. AMPK is at the core of this process as a crucial regulator that senses energy levels within cells. Despite the well-documented advantages of CR, long-term adherence to such a dietary regimens is often impractical for individuals [4]. Therefore, identifying pharmacological agents that could effectively mimic CR effects has emerged as a significant research area within the field of aging science. The presented study positions LCA as an effective analog for CR, offering a promising opportunity for therapeutic intervention.

Based on conventional wisdom, AMPK is primarily activated when energy stores are depleted and glucose levels are decreased [5]. However, the authors discovered that in CR mice, blood glucose did not fall to the levels that would reportedly trigger AMPK activation. This observation indicated that AMPK activation during CR was not directly driven by reduced glucose levels. To further explore this phenomenon, the authors added serum from CR mice to cell cultures and observed sustained AMPK activation even under high glucose concentrations (Figure 1A). This result suggests that a factor in the serum of CR mice can activate AMPK glucose level-independently. The authors conducted a comprehensive metabolomic analysis of the serum from CR mice using various mass spectrometric techniques, and registered significant changes in the abundance of 695 metabolites during CR. Notably, only LCA could recapitulate AMPK activation at physiological concentrations (compared to the control serum) (Figure 1B,C). Furthermore, AMPK activation has been observed in mice as well as in other model organisms, such as nematodes and Drosophila, upon LCA supplementation.

The authors further investigated the signaling pathway by which LCA activates AMPK. They observed that AMPK activation via either LCA or CR did not lead to an increase in AMP or cytosolic calcium levels. This finding ruled out traditional AMP- or calcium-dependent activation mechanisms. The authors explored the lysosomal sensing pathway and found that lithocholic acid failed to activate AMPK when key proteins involved in lysosomal signaling, such as AXIN and LAMTOR1, were knocked out.

The authors also discovered that LCA significantly affected the posttranslational modifications of v-ATPase, particularly its acetylation. By treating with a deacetylase inhibitor that prevented acetylation modification removal on v-ATPase, the authors could block the LCA-mediated activation of AMPK. Using protein modification-based mass spectrometry, the authors identified 263 acetylation sites across all 21 subunits of the v-ATPase complex. Mutating these sites to arginine, a strategy used to mimic deacetylation, has allowed researchers to identify specific sites critical for activation. Notably, introducing triple mutations at three lysine residues (K52, K99, and K191) of the V1E1 subunit of v-ATPase (V1E1-3KR) resulted in v-ATPase inhibition and AMPK activation to a degree comparable to that observed with LCA treatment, even in the absence of starvation. Furthermore, expression of the V1E1-3KR mutation in nematodes, Drosophila, and mouse muscle triggered AMPK activation and delayed aging, demonstrating the broader physiological relevance of this mechanism.

Next, the authors investigated the mechanism by which LCA induces the deacetylation of the V1E1 subunit of v-ATPase. The sirtuin family (SIRT1–SIRT7) and the histone deacetylase (HDAC) family (HDAC1–HDAC11) are the primary deacetylase families in vivo. Sirtuins modulate metabolic processes, including fatty acid oxidation and NAD⁺-dependent enzymatic activity, via deacetylation-mediated posttranslational modifications. These modifications enhance mitochondrial efficiency, attenuate oxidative stress, and extend lifespan across model organisms such as yeast, nematodes, Drosophila, and mice. The authors found that the expression of any member of the sirtuin family in cells led to V1E1 deacetylation, whereas the expression of HDACs did not, suggesting that SIRTs rather than HDACs are responsible for V1E1 deacetylation. To further validate this finding, the authors knocked out all seven sirtuins, which completely abolished both LCA-induced deacetylation of V1E1 and the subsequent activation of AMPK. Thus, LCA activates the lysosomal pathway and stimulates AMPK by enhancing the activity of sirtuins that deacetylate V1E1 and inhibit V-ATPase activity.

The authors sought to further elucidate the mechanism by which LCA promotes SIRT activity in vitro, but found that it failed to activate prokaryotically expressed SIRT1 (contrary to their in vivo findings). To investigate this discrepancy, the components in the in vitro system were progressively replaced with those that were more representative of the intracellular environment. This approach ultimately led to the discovery of an unknown chaperone protein within the cells that binds to LCA, thereby activating SIRT1. To identify this chaperone, the authors incubated SIRT1 with cell lysates for protein profiling and identified 1655 potential interacting proteins. Of these, 157 directly interacted with SIRT1. Further analysis revealed that knockdown of a protein called TULP3 almost completely abolished the activation of AMPK by LCA. Additionally, a TULP3 mutant that does not bind to LCA inhibits the activation of both SIRT1 and AMPK by LCA. These findings suggest that TULP3, through its interaction with lithocholic acid, plays a crucial role in the activation of SIRT1 and initiates a cascade of downstream signaling events(Figure 1D).

In summary, this study identifies LCA as a metabolite that mimics the effects of caloric restriction, and elucidated the mechanisms through which LCA influences aging, particularly the activation of the TULP3-Sirtuin-v-ATPase-AMPK axis. This result addresses a critical gap in our understanding of how the body senses CR-induced metabolic signals and their role in slowing aging. Particularly noteworthy is the finding that LCA bypasses classical AMP/calcium signaling pathways, offering a pharmacological advantage for aging interventions by potentially avoiding the metabolic stress induced by chronic energy deprivation. This bypass mechanism minimizes the adverse effects of prolonged nutrient scarcity and opens up new avenues for more sustainable therapeutic strategies. Moreover, the structural mapping of critical V1E1 acetylation sites(K52/K99/K191) offers precision targets for developing site-specific SIRT activators to enhance deacetylation fidelity, thereby refining the development of precision therapeutics Additionally, the study raises intriguing possibilities regarding the involvement of other LCA receptors in metabolic crosstalk. The potential for these receptors to interact with diverse metabolic pathways could inspire the development of combination therapies that target multiple nodes within the TULP3-Sirtuin-v-ATPase network, amplifying their collective impact on aging and metabolic regulation. Overall, this study provides valuable insights for developing therapeutic interventions aimed at enhancing health and extending the lifespan, while also offering new targets for anti-aging research. These insights may not only contribute to aging interventions but also provide potential breakthroughs in treating age-related diseases through metabolic modulation.

Yiran Wu: visualization (lead), writing – original draft (lead). Zhengyu Gao: visualization, writing – original draft. Long Zhang: writing – review and editing (lead). All authors have read and approved the final manuscript.

The authors have nothing to report.

The authors declare no conflicts of interest.