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This could be explained by the fact that AMPK exists as multiple heterotrimer complexes comprising a catalytic \u03b1-subunit (\u03b11 and \u03b12) and regulatory \u03b2 (\u03b21 and \u03b22)- and \u03b3 (\u03b31, \u03b32, \u03b33)-subunits, which are uniquely distributed across different cell types. There has been keen interest in developing specific and isoform-selective AMPK-activating drugs for therapeutic use and also as research tools. Moreover, establishing ways of enhancing cellular AMPK activity would be beneficial for both purposes. Here, we investigated if a recently described potent AMPK activator called 991, in combination with the commonly used activator 5-aminoimidazole-4-carboxamide riboside or contraction, further enhances AMPK activity and glucose transport in mouse skeletal muscle ex vivo. Given that the \u03b33-subunit is exclusively expressed in skeletal muscle and has been implicated in contraction-induced glucose transport, we measured the activity of AMPK\u03b33 as well as ubiquitously expressed \u03b31-containing complexes. We initially validated the specificity of the antibodies for the assessment of isoform-specific AMPK activity using AMPK-deficient mouse models. We observed that a low dose of 991 (5 \u03bcM) stimulated a modest or negligible activity of both \u03b31- and \u03b33-containing AMPK complexes. Strikingly, dual treatment with 991 and 5-aminoimidazole-4-carboxamide riboside or 991 and contraction profoundly enhanced AMPK\u03b31\/\u03b33 complex activation and glucose transport compared with any of the single treatments. The study demonstrates the utility of a dual activator approach to achieve a greater activation of AMPK and downstream physiological responses in various cell types, including skeletal muscle. <\/jats:p>","DOI":"10.1152\/ajpendo.00237.2016","type":"journal-article","created":{"date-parts":[[2016,8,30]],"date-time":"2016-08-30T22:44:28Z","timestamp":1472597068000},"page":"E706-E719","source":"Crossref","is-referenced-by-count":82,"title":["Benzimidazole derivative small-molecule 991 enhances AMPK activity and glucose uptake induced by AICAR or contraction in skeletal muscle"],"prefix":"10.1152","volume":"311","author":[{"given":"Laurent","family":"Bultot","sequence":"first","affiliation":[{"name":"Nestl\u00e9 Institute of Health Sciences SA, EPFL Innovation Park, Lausanne, Switzerland;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas E.","family":"Jensen","sequence":"additional","affiliation":[{"name":"Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Copenhagen, Denmark;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu-Chiang","family":"Lai","sequence":"additional","affiliation":[{"name":"Universit\u00e9 catholique de Louvain and de Duve Institute, Brussels, Belgium;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Agnete L. 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