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However, current methods rely on overexpressed epitope-tagged GLUT4 constructs or indirect measurements, limiting their physiological relevance and applicability. To overcome these challenges, we developed an innovative high-sensitivity imaging-based method that enables the direct assessment of endogenous GLUT4 translocation in primary skeletal muscle fibers. This approach uses antibodies targeting exofacial epitopes on native GLUT4. Our method allows multiplexed analysis of multiple insulin-sensitive processes, including transferrin receptor trafficking and FOXO nuclear exclusion, alongside mitochondrial oxidative stress. This comprehensive approach provides a unique opportunity to simultaneously assess insulin action across different signaling branches within individual muscle fibers. We validated this method across multiple inbred mouse strains and models of insulin resistance, including chronic insulin exposure, palmitate treatment, and obesity induced by a high-fat diet. Notably, we identified a selective defect in GLUT4 trafficking in insulin-resistant muscle fibers, while other insulin-dependent processes remained intact. By offering a high-fidelity model that maintains physiological relevance, this novel approach represents a significant advancement in the study of skeletal muscle insulin resistance and provides a powerful tool for dissecting gene-environment interactions that underpin metabolic disease.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Article Highlights<\/jats:title>\n                    <jats:p>Insulin-stimulated GLUT4 translocation in skeletal muscle is crucial for whole-body glucose homeostasis, but direct methods to measure endogenous GLUT4 translocation in adult muscle are lacking. We developed a high-sensitivity, high-fidelity imaging method to directly assess endogenous GLUT4 translocation in primary skeletal muscle fibers without the need for overexpressed epitope-tagged constructs. The method enables simultaneous assessment of several makers of insulin sensitivity and mitochondrial oxidative stress within individual muscle fibers. The approach is versatile and was validated across genetically diverse mouse strains and under various insulin resistance conditions.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.2337\/db25-0022","type":"journal-article","created":{"date-parts":[[2025,5,7]],"date-time":"2025-05-07T12:42:05Z","timestamp":1746621725000},"page":"1121-1134","update-policy":"https:\/\/doi.org\/10.2337\/ada-journal-policies","source":"Crossref","is-referenced-by-count":7,"title":["Analysis of Multiple Insulin Actions in Single Muscle Fibers From Insulin-Resistant Mice Reveals Selective Defect in Endogenous GLUT4 Translocation"],"prefix":"10.2337","volume":"74","author":[{"given":"Sebastian","family":"Judge","sequence":"first","affiliation":[{"name":"School of Life and Environmental Sciences, University of Sydney 1 , Camperdown, New South Wales, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stewart W.C.","family":"Masson","sequence":"additional","affiliation":[{"name":"School of Life and Environmental Sciences, University of Sydney 1 , Camperdown, New South Wales, Australia"},{"name":"Charles Perkins Centre, University of Sydney 2 , Camperdown, New South Wales, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"S\u00f8ren","family":"Madsen","sequence":"additional","affiliation":[{"name":"School of Life and Environmental Sciences, University of Sydney 1 , Camperdown, New South Wales, Australia"},{"name":"Charles Perkins Centre, University of Sydney 2 , Camperdown, New South Wales, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meg","family":"Potter","sequence":"additional","affiliation":[{"name":"Charles Perkins Centre, University of Sydney 2 , Camperdown, New South Wales, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5946-5257","authenticated-orcid":false,"given":"David E.","family":"James","sequence":"additional","affiliation":[{"name":"School of Life and Environmental Sciences, University of Sydney 1 , Camperdown, New South Wales, Australia"},{"name":"Charles Perkins Centre, University of Sydney 2 , Camperdown, New South Wales, Australia"},{"name":"School of Medical Sciences, University of Sydney 3 , Camperdown, New South Wales, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6609-6151","authenticated-orcid":false,"given":"James G.","family":"Burchfield","sequence":"additional","affiliation":[{"name":"School of Life and Environmental Sciences, University of Sydney 1 , Camperdown, New South Wales, Australia"},{"name":"Charles Perkins Centre, University of Sydney 2 , Camperdown, New South Wales, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5227-4482","authenticated-orcid":false,"given":"Alexis","family":"Diaz-Vegas","sequence":"additional","affiliation":[{"name":"School of Life and Environmental Sciences, University of Sydney 1 , Camperdown, New South Wales, Australia"},{"name":"Charles Perkins Centre, University of Sydney 2 , Camperdown, New South Wales, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1167","published-online":{"date-parts":[[2025,4,24]]},"reference":[{"key":"2026061113021191400_B1","doi-asserted-by":"crossref","first-page":"751","DOI":"10.1038\/s41580-021-00390-6","article-title":"The aetiology and molecular landscape of insulin resistance","volume":"22","author":"James","year":"2021","journal-title":"Nat Rev Mol Cell Biol"},{"key":"2026061113021191400_B2","doi-asserted-by":"crossref","first-page":"7090","DOI":"10.1016\/S0021-9258(19)68926-X","article-title":"Potential mechanism of insulin action on glucose transport in the isolated rat diaphragm. 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