FoxO Transcription Factors Are Critical Regulators of Diabetes-Related Muscle Atrophy

Insulin deficiency and uncontrolled diabetes lead to a catabolic state with decreased muscle strength, contributing to disease-related morbidity. FoxO transcription factors are suppressed by insulin and thus are key mediators of insulin action. To study their role in diabetic muscle wasting, we created mice with muscle-specific triple knockout of FoxO1/3/4 and induced diabetes in these M-FoxO-TKO mice with streptozotocin (STZ). Muscle mass and myofiber area were decreased 20-30% in STZ-Diabetes mice due to increased ubiquitin-proteasome degradation and autophagy alterations, characterized by increased LC3-containing vesicles, and elevated levels of phosphorylated ULK1 and LC3-II. Both the muscle loss and markers of increased degradation/autophagy were completely prevented in STZ FoxO-TKO mice. Transcriptomic analyses revealed FoxO-dependent increases in ubiquitin-mediated proteolysis pathways in STZ-Diabetes, including regulation of Fbxo32 (Atrogin1), Trim63 (MuRF1), Bnip3L, and Gabarapl. These same genes were increased 1.4- to 3.3-fold in muscle from humans with type 1 diabetes after short-term insulin deprivation. Thus, FoxO-regulated genes play a rate-limiting role in increased protein degradation and muscle atrophy in insulin-deficient diabetes. Muscle-specific FoxO1, FoxO3, and FoxO4 triple knockout (M-FoxO-TKO) mice were generated using ACTA1-Cre (stock number 006149; The Jackson Laboratory) and FoxO1/3/4 triple floxed mice, provided by Dr. Domenico Accili, Colombia University Medical Center. Littermate controls were used for all experiments, as the mice are on a mixed background containing C57Blk6, C57Blk6J, and 129 strains. For STZ treatments, mice were fasted overnight and then injected intraperitoneally with a single high dose of STZ (150 mg/kg) (S0130; Sigma) dissolved in 100 mmol/L citrate buffer (pH 4.5) or injected with citrate buffer alone as a control, then immediately given access to food. Female mice were given a 150 mg/kg dose followed by a 75 mg/kg dose of STZ on day 3, since female mice are less responsive to STZ (24). Mice were monitored for hyperglycemia on days 3, 7, 12, and 15, and only mice in which random blood glucose remained >300 mg/dL for the following 9–12 days were used for experiments.