Low-dose metformin targets the lysosomal AMPK pathway through PEN2

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Metformin is a widely prescribed antidiabetic medication with known anti-aging and anti-cancer properties.
• The precise molecular target and mechanism of action for metformin at clinical doses, particularly regarding AMP-activated protein kinase (AMPK) activation, remain incompletely understood.

Purpose of the Study:

• To identify the direct molecular target of metformin.
• To elucidate the mechanism by which metformin activates AMP-activated protein kinase (AMPK).
• To investigate the role of identified targets in metformin's therapeutic effects.

Main Methods:

• Synthesis of a photoactive metformin probe to identify binding partners.
• Biochemical assays to determine binding affinity (dissociation constant).
• Genetic manipulation (knockout and mutant studies) in cell lines and model organisms (C. elegans).
• In vivo studies assessing metabolic effects (hepatic fat content, glucose lowering) and lifespan extension.

Main Results:

• Metformin was found to directly inhibit the lysosomal proton pump v-ATPase.
• PEN2, a subunit of γ-secretase, was identified as a metformin binding partner.
• Metformin-bound PEN2 forms a complex with ATP6AP1, a v-ATPase subunit, leading to v-ATPase inhibition and AMPK activation.
• PEN2 knockout or non-binding mutants abolished metformin-induced AMPK activation.
• In vivo, PEN2 is crucial for metformin's effects on hepatic fat reduction and glucose lowering, as well as lifespan extension in C. elegans.

Conclusions:

• Metformin binds to PEN2, initiating a signaling cascade via ATP6AP1 that inhibits v-ATPase and activates AMPK.
• This mechanism explains metformin's therapeutic benefits, including anti-diabetic, anti-aging, and anti-cancer effects, by linking it to the lysosomal glucose-sensing pathway.
• Targeting the PEN2-ATP6AP1-v-ATPase axis provides a molecular basis for metformin's pleiotropic actions and suggests potential therapeutic strategies.