Low-dose metformin targets the lysosomal AMPK pathway through PEN2

<jats:title>Abstract</jats:title><jats:p>Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects<jats:sup>1–4</jats:sup>. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action<jats:sup>4,5</jats:sup>; 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 starvation<jats:sup>6</jats:sup>. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase<jats:sup>7</jats:sup>, 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-ATPase<jats:sup>8</jats:sup>, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of <jats:italic>PEN2</jats:italic> or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of <jats:italic>Pen2</jats:italic> abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of <jats:italic>Pen2</jats:italic> impairs its glucose-lowering effects. Furthermore, knockdown of <jats:italic>pen-2</jats:italic> in <jats:italic>Caenorhabditis elegans</jats:italic> 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.</jats:p>