Low-dose metformin targets the lysosomal AMPK pathway through PEN2

Teng Ma,Xiao Tian,Xiaoyan Wei,Jin-Wei Feng,Shu-Yong Lin,Yaying Wu,Changchuan Xie,Xianming Deng,Weiping Jia,Yongsheng Yu ,Zhiyun Ye,Xi Huang,Wen Wang,Mengqi Li,Lei Zhang,Shating Long,Jianfeng Wu,Hai‐Long Piao ,Chunyan Yang,Cixiong Zhang,Yaxin Yu,Qi Qu,Zhichao Wang ,Luming Yao,Mingliang Zhang,Baoding Zhang,Yu Wang,Maoshu Zhu ,Chun Li,Zheni Xu,Junjie Chen,Ying He,Yuqian Bao,Chen-Song Zhang

doi:10.1038/s41586-022-04431-8

Abstract

Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1–4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.

Highlights

Metformin is the usual first-line drug of choice to reduce blood glucose levels in patients with type 2 diabetes mellitus
We previously reported that glucose deprivation can activate lysosomal AMPK without increasing AMP/ADP levels through v-ATPase, Ragulator and AXIN24, which are downstream of the fructose-1,6-bisphosphate sensor aldolase
The PEN2–ATP6AP1 axis constitutes a signalling shunt that intersects the lysosomal v-ATPase–AXIN–AMPK axis, which enables metformin at low concentration to make use of the AMP-independent AMPK-activation pathway, which is can be triggered by glucose starvation

Summary

Introduction

Metformin is the usual first-line drug of choice to reduce blood glucose levels in patients with type 2 diabetes mellitus. We found that metformin at clinical doses sufficiently inhibited the vacuolar H+-ATPase (v-ATPase) on the lysosome (Fig. 1a, b and Extended Data Fig. 1, with detailed discussions in Supplementary Note 1). We observed that depletion of PEN2, but not others, rendered the cells insensitive to metformin treatment, as assessed by levels of AMPK activation and inhibition of v-ATPase Knockout of PEN2 blocked low-dose metformin-induced AMPK activation and v-ATPase inhibition in primary hepatocytes, MEFs and HEK293T cells (Fig. 2b, c and Extended Data Fig. 3c–i, k, l; note that knockout of PEN2 did not affect basal lysosomal pH levels (Supplementary Note 2)). 4f, g and 5a), and metformin did not alter the subcellular localization of PEN2 (Extended Data Fig. 5b, c) These results indicate that the pool of lysosomally localized PEN2 may have a distinct role, whereby it participates in metformin-induced AMPK activation (discussed in Supplementary Note 5). Constructs of PEN2 fused to other organelle-specific proteins did not restore AMPK activation by metformin when re-introduced into Pen2–/– MEFs (Extended Data Fig. 5d, with validation data in Extended Data Fig. 5d, e)

Methods

Results

Conclusion