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

Biswas, D.; Espino-Gonzalez, E.; Ahwazi, D.; Freemantle, J. B.; Jomard, C.; Brorson, J.; Schou, A. N.; Farup, J.; GONDIN, J.; Just, J.; Foretz, M.; Treebak, J. T.; Agerholm, M.; Sakamoto, K.

doi:10.1101/2025.09.26.678768

bioRxiv · 10.1101/2025.09.26.678768

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

Biswas, D.·Espino-Gonzalez, E.·Ahwazi, D.·Freemantle, J. B.·Jomard, C.·Brorson, J.·Schou, A. N.·Farup, J.·GONDIN, J.·Just, J.·Foretz, M.·Treebak, J. T.·Agerholm, M.·Sakamoto, K.

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Abstract

ObjectiveSmall-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 AMPK{gamma} subunit plays a central role in energy sensing. While the skeletal muscle-selective {gamma}3 isoform is essential for AMP/ZMP-induced glucose uptake, it is dispensable for ADaM site-binding activators. We hypothesized that the predominant {gamma}1 isoform is required for ADaM site activator-stimulated glucose uptake in skeletal muscle. MethodsSingle-nucleus RNA sequencing (snRNA-seq) was performed on mouse and human skeletal muscle mapping AMPK subunit isoform distribution across resident cell types. To determine {gamma} isoform-specific requirements for activator-stimulated glucose uptake, skeletal muscle-specific inducible AMPK{gamma}1/{gamma}3 double knockout (im{gamma}1-/-/{gamma}3-/-) and single knockout (im{gamma}1-/- and im{gamma}3-/-) 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. ResultssnRNA-seq revealed distinct AMPK isoform distribution: {gamma}1 was ubiquitously expressed, whereas {gamma}3 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 im{gamma}1-/-/{gamma}3-/- 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 im{gamma}3-/-, whereas both activators fully retained effects on glucose uptake and glucose lowering in im{gamma}1-/- mice. ConclusionsWhile {gamma}1 predominates in stabilizing the AMPK2{beta}2{gamma}1 complex, it is dispensable for AMPK activator-stimulated glucose uptake in skeletal muscle, whether mediated via the nucleotide-binding or ADaM site.

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Biswas, D., Espino-Gonzalez, E., Ahwazi, D., Freemantle, J. B., Jomard, C., Brorson, J., Schou, A. N., Farup, J., GONDIN, J., Just, J., Foretz, M., Treebak, J. T., Agerholm, M., Sakamoto, K.. 2025-09-29. Common and distinct roles of AMPKγ isoforms in small-molecule activator-stimulated glucose uptake in mouse skeletal muscle. https://doi.org/10.1101/2025.09.26.678768

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