AMPKα2 isoform mediates mTORC2 activation by glucose starvation in endothelial cells

Abstract

AMP-activated protein kinase (AMPK) and the mechanistic target of rapamycin (mTOR) are central regulators of cellular metabolism. While AMPK is known to inhibit mTOR complex 1 (mTORC1), its role in regulating mTORC2 remains enigmatic, with recent evidence suggesting context-dependent activation. Here, we investigated the specific roles of AMPK catalytic isoforms in coordinating mTOR signaling and functional metabolic adaptations in human umbilical vein endothelial cells (HUVECs) during glucose starvation. We found that loss of the major AMPK1 isoform triggers an increase in otherwise minor AMPK2 expression. While both isoforms are activated by glucose starvation and phosphorylate the canonical substrate acetyl-CoA carboxylase (ACC), only AMPK2 is necessary and sufficient for transient activation of the mTORC2 signaling, as monitored by phosphorylation of downstream reporters, Akt and serum/glucocorticoid regulated kinase 1 (SGK1). This AMPK2-dependent mTORC2 activation peaked at 30 minutes of starvation and then declined under prolonged starvation stress. Surprisingly, genetic ablation of the AMPK2-mTORC2 axis through knockdown of AMPK2, both AMPK isoforms, or the essential mTORC2 component Rictor failed to attenuate the starvation-induced compensatory increase in glucose uptake. This adaptive response occurred robustly and identically across all genetic backgrounds. Collectively, our findings reveal an isoform-specific signaling module wherein AMPK2 transiently activates mTORC2, but this pathway is functionally uncoupled from the critical adaptive response of glucose uptake, thus uncovering a remarkable resilience in the endothelial metabolic network and indicating that other, parallel pathways are the primary drivers of this essential survival mechanism.