Common and distinct roles of AMPKγ isoforms in small-molecule activator-stimulated glucose uptake in mouse skeletal muscle

ABSTRACT Objective Small-molecule activators targeting the allosteric drug and metabolite (ADaM) site of AMPK enhance insulin-independent glucose uptake in skeletal muscle and lower glucose in preclinical models of hyperglycemia. The regulatory AMPKγ subunit plays a central role in energy sensing. While the skeletal muscle-selective γ3 isoform is essential for AMP/ZMP-induced glucose uptake, it is dispensable for ADaM site-binding activators. We hypothesized that the predominant γ1 isoform is required for ADaM site activator-stimulated glucose uptake in skeletal muscle. Methods Single-nucleus RNA sequencing (snRNA-seq) was performed on mouse and human skeletal muscle mapping AMPK subunit isoform distribution across resident cell types. To determine γ isoform-specific requirements for activator-stimulated glucose uptake, skeletal muscle-specific inducible AMPKγ1/γ3 double knockout (imγ1 -/- /γ3 -/- ) and single knockout (imγ1⁻ / ⁻ and imγ3⁻ / ⁻) mice were generated . Ex vivo glucose uptake was measured following treatment with AICAR (AMP-mimetic) or MK-8722 (ADaM site activator), and in vivo MK-8722-induced blood glucose lowering was assessed. Results snRNA-seq revealed distinct AMPK isoform distribution: γ1 was ubiquitously expressed, whereas γ3 was enriched in glycolytic myofibers in both mouse and human skeletal muscle. Ex vivo , glucose uptake stimulated by either AICAR or MK-8722 was abolished in imγ1⁻ / ⁻/γ3⁻ / ⁻ muscle, and MK-8722-induced blood glucose lowering was significantly blunted in vivo . AICAR but not MK-8722-stimulated muscle glucose uptake was abolished in imγ3⁻ / ⁻, whereas both activators fully retained effects on glucose uptake and glucose lowering in imγ1⁻ / ⁻ mice. Conclusions While γ1 predominates in stabilizing the AMPKα2β2γ1 complex, it is dispensable for AMPK activator-stimulated glucose uptake in skeletal muscle, whether mediated via the nucleotide-binding or ADaM site.