3035-LB: Hypothalamic Rap1-AMPK Signaling Axis Mediates the Antidiabetic Actions of Metformin

Introduction and Objective: The brain regulates whole-body glucose homeostasis. We previously identified hypothalamic small GTPase Rap1 as a key regulator of hyperglycemia in diet-induced obesity. Forebrain Rap1-deficient mice are unresponsive to metformin, indicating VMH Rap1 signaling mediates metformin action. To elucidate the underlying molecular mechanism, we investigated the brain Rap1 signaling pathway mediating antidiabetic action of metformin. Methods: BioID proteomics identified central AMP-activated protein kinase (AMPK) as a downstream Rap1 target. To validate this target, we generated brain-specific AMPK knockout mice and assessed their response to metformin by measuring blood glucose and glucose tolerance. Furthermore, we examined AMPK phosphorylation levels via Western Blotting and Immunofluorescence in control and central Rap1-deficient mice following vehicle or metformin administration. Results: Our BioID data suggested a physical proximity between Rap1 and AMPK-anchoring proteins, which suggests a novel neural pathway. Moreover, we found that metformin treatment significantly increased AMPK phosphorylation in the hypothalamus, specifically in the VMH (fold change 2.26, p<0.01). More importantly, the glucose-lowering effect of metformin was abolished in neuronal AMPK-deficient mice (p=0.8813), indicating that central AMPK is required for its therapeutic effect. Furthermore, genetic deletion of central Rap1 leads to constitutive activation of AMPK, as evidenced by increased phosphorylation levels in Rap1-deficient mice compared to controls. This suggests the hypothalamic Rap1-AMPK signaling axis mediated the effect of metformin. Conclusion: Collectively, our findings indicate that metformin exerts its antidiabetic effects by targeting the hypothalamic Rap1-AMPK signaling axis. This study not only uncovers the detailed neural mechanism of metformin action but also highlights the central Rap1-AMPK pathway as a potential therapeutic target for diabetes treatment. Disclosure W. Lu: None. H. Kosako: None. M. Fukuda: None.