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In the canonical activation mechanism, it is activated by increases in AMP:ATP and ADP:ATP ratios that signify declining cellular energy status. Once activated, AMPK phosphorylates numerous targets that promote catabolic pathways generating ATP, while inhibiting anabolic and other processes that consume ATP, thus acting to restore energy homeostasis. Pharmacological agents that activate AMPK have been useful in identifying downstream targets and have potential as drugs for treatment of metabolic disorders such as Type 2 diabetes and non-alcoholic fatty liver disease. One such agent is C13, a pro-drug with a phosphonate bis(isobutyryloxymethyl) ester moiety, with the isobutyryloxymethyl groups increasing membrane permeability. Following cellular uptake, C13 is cleaved to release C2, an AMP analogue and potent AMPK activator that is specific for complexes containing the \u03b11 (but not the \u03b12) catalytic subunit isoform. This has previously been assumed to be the sole mechanism by which C13 activates AMPK, with potential roles for the isobutyryloxymethyl groups being ignored. We now report that, following cleavage from C13, these protective groups are metabolized to formaldehyde, an agent that inhibits mitochondrial function and increases cellular AMP:ATP ratios, thus providing additional AMPK activation by the canonical mechanism.<\/jats:p>","DOI":"10.1042\/bcj20240425","type":"journal-article","created":{"date-parts":[[2024,9,2]],"date-time":"2024-09-02T14:21:51Z","timestamp":1725286911000},"page":"1203-1219","update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["The pro-drug C13 activates AMPK by two distinct mechanisms"],"prefix":"10.1042","volume":"481","author":[{"given":"Jordana B.","family":"Freemantle","sequence":"first","affiliation":[{"name":"1Division of Cell Signalling and Immunology, Sir James Black Centre, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dinesh","family":"Shah","sequence":"additional","affiliation":[{"name":"1Division of Cell Signalling and Immunology, Sir James Black Centre, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dylan M.","family":"Lynch","sequence":"additional","affiliation":[{"name":"2Centre for Targeted Protein Degradation, School of Life Sciences, University of Dundee, 1 James Lindsay Place, Dundee DD1 5JJ, U.K."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8654-1670","authenticated-orcid":false,"given":"Alessio","family":"Ciulli","sequence":"additional","affiliation":[{"name":"2Centre for Targeted Protein Degradation, School of Life Sciences, University of Dundee, 1 James Lindsay Place, Dundee DD1 5JJ, U.K."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Harinder S.","family":"Hundal","sequence":"additional","affiliation":[{"name":"1Division of Cell Signalling and Immunology, Sir James Black Centre, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8373-7379","authenticated-orcid":false,"given":"D. 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