Covert, Jacob D.Grice, Brian A.Thornburg, Matthew G.Kaur, ManpreetRyan, Andrew P.Tackett, LixuanBhamidipati, ThejaStull, Natalie D.Kim, TeayounHabegger, Kirk M.McClain, Donald A.Brozinick, Joseph T.Elmendorf, Jeffrey S.2023-11-292023-11-292023Covert JD, Grice BA, Thornburg MG, et al. An early, reversible cholesterolgenic etiology of diet-induced insulin resistance. Mol Metab. 2023;72:101715. doi:10.1016/j.molmet.2023.101715https://hdl.handle.net/1805/37198Objective: A buildup of skeletal muscle plasma membrane (PM) cholesterol content in mice occurs within 1 week of a Western-style high-fat diet and causes insulin resistance. The mechanism driving this cholesterol accumulation and insulin resistance is not known. Promising cell data implicate that the hexosamine biosynthesis pathway (HBP) triggers a cholesterolgenic response via increasing the transcriptional activity of Sp1. In this study we aimed to determine whether increased HBP/Sp1 activity represented a preventable cause of insulin resistance. Methods: C57BL/6NJ mice were fed either a low-fat (LF, 10% kcal) or high-fat (HF, 45% kcal) diet for 1 week. During this 1-week diet the mice were treated daily with either saline or mithramycin-A (MTM), a specific Sp1/DNA-binding inhibitor. A series of metabolic and tissue analyses were then performed on these mice, as well as on mice with targeted skeletal muscle overexpression of the rate-limiting HBP enzyme glutamine-fructose-6-phosphate-amidotransferase (GFAT) that were maintained on a regular chow diet. Results: Saline-treated mice fed this HF diet for 1 week did not have an increase in adiposity, lean mass, or body mass while displaying early insulin resistance. Consistent with an HBP/Sp1 cholesterolgenic response, Sp1 displayed increased O-GlcNAcylation and binding to the HMGCR promoter that increased HMGCR expression in skeletal muscle from saline-treated HF-fed mice. Skeletal muscle from these saline-treated HF-fed mice also showed a resultant elevation of PM cholesterol with an accompanying loss of cortical filamentous actin (F-actin) that is essential for insulin-stimulated glucose transport. Treating these mice daily with MTM during the 1-week HF diet fully prevented the diet-induced Sp1 cholesterolgenic response, loss of cortical F-actin, and development of insulin resistance. Similarly, increases in HMGCR expression and cholesterol were measured in muscle from GFAT transgenic mice compared to age- and weight-match wildtype littermate control mice. In the GFAT Tg mice we found that these increases were alleviated by MTM. Conclusions: These data identify increased HBP/Sp1 activity as an early mechanism of diet-induced insulin resistance. Therapies targeting this mechanism may decelerate T2D development.en-USAttribution 4.0 InternationalCholesterolInsulin resistanceMembraneSkeletal muscleArticle