Background Muscle mitochondrial decline is associated with aging‐related muscle weakness and insulin resistance. FoxO transcription factors are targets of insulin action and deletion of FoxOs improves mitochondrial function in diabetes. However, disruptions in proteostasis and autophagy are hallmarks of aging and the effect of chronic inhibition of FoxOs in aged muscle is unknown. This study investigated the role of FoxOs in regulating muscle strength and mitochondrial function with age. Methods We measured muscle strength, cross‐sectional area, muscle fibre‐type, markers of protein synthesis/degradation, central nuclei, glucose/insulin tolerance, and mitochondrial bioenergetics in 4.5‐month (Young) and 22–24‐month‐old (Aged) muscle‐specific FoxO1/3/4 triple KO (TKO) and littermate control (Ctrl) mice. Results Lean mass was increased in Aged TKO compared with both Aged Ctrl and younger groups by 26–33% (P < 0.01). Muscle strength, measured by max force of tibialis anterior (TA) contraction, was 20% lower in Aged Ctrl compared with Young Ctrls (P < 0.01) but was not decreased in Aged TKOs. Increased muscle strength in Young and Aged TKO was associated with 18–48% increased muscle weights compared with Ctrls (P < 0.01). Muscle cross‐sectional analysis of TA, soleus, and plantaris revealed increases in fibre size distribution and a 2.5–10‐fold increase in central nuclei in Young and Aged TKO mice, without histologic signs of muscle damage. Age‐dependent increases in Gadd45a and Ube4a expression as well accumulation of K48 polyubiquitinated proteins were observed in quad and TA but were prevented by FoxO deletion. Young and Aged TKO muscle showed minimal changes in autophagy flux and no accumulation of autophagosomes compared with Ctrl groups. Increased strength in Young and Aged TKO was associated with a 10–20% increase in muscle mitochondrial respiration using glutamate/malate/succinate compared with controls (P < 0.05). OXPHOS subunit expression and complex I activity were decreased 16–34% in Aged Ctrl compared with Young Ctrl but were prevented in Aged TKO. Both Aged Ctrl and Aged TKO showed impaired glucose tolerance by 33% compared to young groups (P < 0.05) indicating improved strength and mitochondrial respiration are not due to improved glycemia. Conclusions FoxO deletion increases muscle strength even during aging. Deletion of FoxOs maintains muscle strength in part by mild suppression of atrophic pathways, including inhibition of Gadd45a and Ube4a expression, without accumulation of autophagosomes in muscle. Deletion of FoxOs also improved mitochondrial function by maintenance of OXPHOS in both young and aged TKO.

Uncontrolled diabetes impairs muscle strength and mitochondrial metabolism, leading to increased mortality. FoxOs are key mediators of insulin signaling and we demonstrated that FoxOs mediate muscle atrophy and mitochondrial dysfunction in mice with streptozotocin (STZ) diabetes. Unfortunately, FoxOs are also tumor suppressors and knockout of FoxOs in proliferative tissues causes cancer. We aimed to investigate FoxO targets restricted to skeletal muscle in hopes of improving strength in diabetic conditions. RNA-seq from STZ mice with/without FoxO1/3/4 triple knockout showed several diabetes-induced FoxO targets, with the most robust being LRRC2 (Leucine rich repeating containing 2). The function of LRRC2 is unknown, but gene expression databases show LRRC2 is restricted to muscle and heart, and LRRC2 is enriched in human heart with dilated cardiomyopathy. Adenoviral overexpression of LRRC2 in C2C12 cells localized to mitochondria and showed increase mRNA levels of biogenesis marker PGC1α, and antioxidant genes SOD2 and Gpx3. However, LRRC2 overexpression did not change basal respiration measured in Seahorse. To determine LRRC2's effect on exercise tolerance and mitochondrial bioenergetics in vivo, we created muscle specific LRRC2 knockout (LKO) mice and rendered them diabetic with STZ. Diabetic LKO mice did worse than their littermate controls on treadmill exercise to exhaustion. Soleus permeabilized fibers showed significantly decreased ATP production that was uncoupled from respiration with glutamate/malate. Interestingly, soleus muscles from LKO mice were significantly larger than their littermate controls, whereas extensor digitorum longus and quadriceps were smaller in the diabetic LKO mice. Lastly, mRNA expression of mitochondrial subunits was not changed and thus did not explain the mitochondrial defects. In conclusion, LRRC2 is a novel FoxO target that helps preserve mitochondrial function and exercise tolerance in a Type 1 diabetes mouse model. Disclosure C. M. Penniman: None. G. Bhardwaj: None. C. Nowers: None. B. T. O'neill: Stock/Shareholder; Bristol-Myers Squibb Company, Pfizer Inc., Johnson & Johnson. Funding U.S. Department of Veterans Affairs (lO1BXOO4468)