UMMS Affiliation

Division of Cardiovascular Medicine, Department of Medicine; Graduate School of Biomedical Sciences

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Document Type



Biochemical Phenomena, Metabolism, and Nutrition | Exercise Physiology | Exercise Science | Musculoskeletal System


OBJECTIVE: The immediate signals that couple exercise to metabolic adaptations are incompletely understood. Nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) produces reactive oxygen species (ROS) and plays a significant role in metabolic and vascular adaptation during stress conditions. Our objective was to determine the role of Nox4 in exercise-induced skeletal muscle metabolism.

METHODS: Mice were subjected to acute exercise to assess their immediate responses. mRNA and protein expression responses to Nox4 and hydrogen peroxide (H2O2) were measured by qPCR and immunoblotting. Functional metabolic flux was measured via ex vivo fatty acid and glucose oxidation assays using (14)C-labeled palmitate and glucose, respectively. A chronic exercise regimen was also utilized and the time to exhaustion along with key markers of exercise adaptation (skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase activity) were measured. Endothelial-specific Nox4-deficient mice were then subjected to the same acute exercise regimen and their subsequent substrate oxidation was measured.

RESULTS: We identified key exercise-responsive metabolic genes that depend on H2O2 and Nox4 using catalase and Nox4-deficient mice. Nox4 was required for the expression of uncoupling protein 3 (Ucp3), hexokinase 2 (Hk2), and pyruvate dehydrogenase kinase 4 (Pdk4), but not the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1alpha). Global Nox4 deletion resulted in decreased UCP3 protein expression and impaired glucose and fatty acid oxidization in response to acute exercise. Furthermore, Nox4-deficient mice demonstrated impaired adaptation to chronic exercise as measured by the time to exhaustion and activity of skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase. Importantly, mice deficient in endothelial-Nox4 similarly demonstrated attenuated glucose and fatty acid oxidation following acute exercise.

CONCLUSIONS: We report that H2O2 and Nox4 promote immediate responses to exercise in skeletal muscle. Glucose and fatty acid oxidation were blunted in the Nox4-deficient mice post-exercise, potentially through regulation of UCP3 expression. Our data demonstrate that endothelial-Nox4 is required for glucose and fatty acid oxidation, suggesting inter-tissue cross-talk between the endothelium and skeletal muscle in response to exercise.


Endothelium, Exercise, Metabolic adaptation, Nox4, ROS, Skeletal muscle metabolism

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Copyright © 2021 The Authors. This is an open access article under the CC BY-NC-ND license (

DOI of Published Version



Specht KS, Kant S, Addington AK, McMillan RP, Hulver MW, Learnard H, Campbell M, Donnelly SR, Caliz AD, Pei Y, Reif MM, Bond JM, DeMarco A, Craige B, Keaney JF Jr, Craige SM. Nox4 mediates skeletal muscle metabolic responses to exercise. Mol Metab. 2021 Mar;45:101160. doi: 10.1016/j.molmet.2020.101160. Epub 2021 Jan 2. PMID: 33400973; PMCID: PMC7856463. Link to article on publisher's site

Journal/Book/Conference Title

Molecular metabolism


Full author list omitted for brevity. For the full list of authors, see article.

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Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.