Objective Reactive oxygen species (ROS) have been proposed as signaling molecules mediating exercise training adaptation, but the ROS source has remained unclear. This study aimed to investigate if increased NADPH oxidase (NOX)2-dependent activity during exercise is required for long-term high-intensity interval training (HIIT) in skeletal muscle using a mouse model lacking functional NOX2 complex due to absent p47phox ( Ncf1 ) subunit expression ( ncf1* mutation). Methods HIIT was investigated after an acute bout of exercise and after a chronic intervention (3x/week for 6 weeks) in wild-type (WT) vs. NOX2 activity-deficient ( ncf1* ) mice. NOX2 activation during HIIT was measured using an electroporated genetically-encoded biosensor. Immunoblotting and single-fiber microscopy was performed to measure classical exercise-training responsive endpoints in skeletal muscle. Results A single bout of HIIT increased NOX2 activity measured as p47-roGFP oxidation immediately after exercise but not 1 h or 4 h after exercise. After a 6-week HIIT regimen, improvements in maximal running capacity and some muscle training-markers responded less to HIIT in the ncf1* mice compared to WT, including superoxide dismutase 2, catalase, hexokinase II, pyruvate dehydrogenase and protein markers of mitochondrial oxidative phosphorylation complexes. Strikingly, HIIT-training increased mitochondrial network area and decreased fragmentation in WT mice only. Conclusion This study suggests that HIIT exercise increases NOX2 activity in skeletal muscle and shows that NOX2 activity is required for specific skeletal muscle adaptations to HIIT relating to antioxidant defense, glucose metabolism, and mitochondria.

Cancer treatment evokes impediments to liver metabolism that culminate in fatty liver. This study determined hepatic fatty acid composition and expression of genes and mediators involved in lipid metabolism following chemotherapy treatment. Female rats bearing the Ward colon tumor were administered Irinotecan (CPT-11) +5-fluorouracil (5-FU) and maintained on a control diet or a diet containing eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) (2.3 g/100 g fish oil). Healthy animals provided with a control diet served as a reference group. Livers were collected one week after chemotherapy. Triacylglycerol (TG), phospholipid (PL), ten lipid metabolism genes, leptin, and IL-4 were measured. Chemotherapy increased TG content and reduced EPA content in the liver. Expression of SCD1 was upregulated by chemotherapy, while dietary fish oil downregulated its expression. Dietary fish oil down-regulated expression of the fatty acid synthesis gene FASN, while restoring the long chain fatty acid converting genes FADS2 and ELOVL2, and genes involved in mitochondrial β-oxidation (CPT1α) and lipid transport (MTTP1), to values similar to reference animals. Neither leptin nor IL-4 were affected by chemotherapy or diet. Depletion of EPA is associated with pathways evoking enhanced TG accumulation in the liver. Restoring EPA through diet may pose a dietary strategy to attenuate chemotherapy-associated impediments in liver fatty acid metabolism.

22 23 GRAPHICAL ABSTRACT 24 25 26 Highlights: 27 • Acute HIIE induces transient NOX2 complex activity in vivo in muscle 28 • Skeletal muscle adaptations to HIIT were impaired in ncf1-deficient mice 29 • Functional NOX2 is necessary for HIIT-induced increased expression of antioxidants enzymes 30 • Ncf1-deficient mice lack HIIT-induced mitochondrial adaptations 31 32 33 34 35 36 37 38 ABSTRACT 39 Objective: Reactive oxygen species (ROS) have been proposed as signaling molecules mediating 40 exercise training adaptation, but the ROS source has remained unclear. This study aimed to 41 investigate the requirement for NADPH oxidase (NOX)2-dependent redox changes induced by acute 42 and long-term high-intensity interval training (HIIT) in skeletal muscle in a mouse model lacking 43 functional NOX2 complex due to deficient p47phox (Ncf1) subunit expression (ncf1* mutation). 44 Methods: HIIT was investigated after an acute bout of exercise and after a chronic intervention (3x 45week for 6 weeks) in wildtype (WT) vs. NOX2 activity-deficient (ncf1*) mice. NOX2 activation 46 during HIIT was measured using a genetically-encoded biosensor. Immunoblotting and single-fiber 47 staining were performed to measure classical exercise-training responsive endpoints in skeletal 48 muscle. 49Results: A single bout of HIIT increased NOX2 activity measured using electroporated p47roGFP 50 oxidation immediately after exercise but not 1h after exercise. After a 6-week of HIIT regime, 51 improvements in maximal running capacity and some muscle training-markers responded less to HIIT 52 in the ncf1* mice compared to WT, including superoxide dismutase (SOD)2, catalase, hexokinase II 53 (HK II), pyruvate dehydrogenase (PDH) and protein markers of mitochondrial oxidative 54 phosphorylation complexes. Strikingly, HIIT-training increased mitochondrial network area and 55 decreased fragmentation in WT mice only. 56 Conclusion:This study provided evidence that HIIT exercise activates NOX2 complex in skeletal 57 muscle and that the presence of functional NOX2 is required for specific skeletal muscle adaptations 58 to HIIT relating to antioxidant defense, glucose metabolism, and mitochondria. 59 60