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. Overall design: 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.

胰岛素缺乏与血糖未受控的糖尿病会引发分解代谢状态，伴随肌肉力量下降，进而增加疾病相关的发病风险。叉头框O（FoxO）转录因子可被胰岛素抑制，因此是胰岛素作用的关键介导因子。为探究其在糖尿病性肌肉消耗中的作用，我们构建了肌肉特异性FoxO1/3/4三基因敲除小鼠（M-FoxO-TKO），并通过链脲佐菌素（streptozotocin, STZ）诱导该模型小鼠患上糖尿病。链脲佐菌素诱导的糖尿病（STZ-Diabetes）小鼠模型中，肌肉质量与肌纤维面积下降20%~30%，这是由于泛素-蛋白酶体降解增强与自噬异常所致，具体表现为含LC3的囊泡增多，以及磷酸化ULK1与LC3-II的水平升高。在STZ处理的FoxO三基因敲除小鼠中，肌肉丢失以及降解/自噬增强的标志物均完全得到抑制。转录组学分析显示，在STZ诱导的糖尿病小鼠中，泛素介导的蛋白水解通路出现FoxO依赖性的激活，其中包括对Fbxo32（Atrogin1）、Trim63（MuRF1）、Bnip3L以及Gabarapl的调控。在短期胰岛素缺乏的1型糖尿病患者肌肉组织中，上述基因的表达量同样升高了1.4~3.3倍。因此，受FoxO调控的基因在胰岛素缺乏型糖尿病的蛋白降解增强与肌肉萎缩过程中发挥限速作用。整体实验设计：肌肉特异性FoxO1、FoxO3及FoxO4三基因敲除（M-FoxO-TKO）小鼠通过ACTA1-Cre工具鼠（品系编号006149，购自杰克逊实验室）与由哥伦比亚大学医学中心Domenico Accili博士馈赠的FoxO1/3/4三基因floxed小鼠构建获得。所有实验均使用同窝对照小鼠，因该小鼠群体的遗传背景为混合背景，包含C57Blk6、C57Blk6J与129品系。STZ造模时，小鼠先经一夜禁食，随后腹腔注射溶于100 mmol/L柠檬酸盐缓冲液（pH 4.5）的高剂量STZ（150 mg/kg，货号S0130，西格玛公司），对照组仅注射等量柠檬酸盐缓冲液，注射后即刻恢复喂食。由于雌性小鼠对STZ的响应性较弱，雌性小鼠先接受150 mg/kg剂量的STZ注射，并于第3天追加75 mg/kg剂量的STZ（24）。分别于第3、7、12、15天监测小鼠的高血糖情况，仅选取后续9~12天内随机血糖持续高于300 mg/dL的小鼠用于后续实验。