Supplementary Material for: Time-Restricted Feeding Restored Insulin-Growth Hormone Balance and Improved Substrate and Energy Metabolism in MC4RKO Obese Mice

<b><i>Background:</i></b> Dysregulation of metabolic regulatory hormones often occurs during the progress of obesity. Key regulatory hormone insulin-growth hormone (GH) balance has recently been proposed to maintain metabolism profiles. Time-restricted feeding (TRF) is an effective strategy against obesity without detailed research on pulsatile GH releasing patterns. <b><i>Methods:</i></b> TRF was performed in an over-eating melanocortin 4 receptor-knockout (MC4RKO) obese mouse model using normal food. Body weight and food intake were measured. Series of blood samples were collected for 6-h pulsatile GH profile, glucose tolerance test, and insulin tolerance test at 5, 8, and 9 weeks of TRF, respectively. Indirect calorimetric recordings were performed by the Phenomaster system at 6 weeks for 1 week, and body composition was measured by nuclear magnetic resonance spectroscopy (NMR). Substrate- and energy metabolism-related gene expressions were measured in terminal liver and subcutaneous white adipose tissues. <b><i>Results:</i></b> TRF increased pulsatile GH secretion in dark phase and suppressed hyperinsulinemia in MC4RKO obese mice to reach a reduced insulin/GH ratio. This was accompanied by the improvement in insulin sensitivity, metabolic flexibility, glucose tolerance, and decreased glucose fluctuation, together with appropriate modification of gene expression involved in substrate metabolism and adipose tissue browning. NMR measurement showed that TRF decreased fat mass but increased lean mass. Indirect calorimeter recording indicated that TRF decreased the respiratory exchange ratio (RER) reflecting consumption of more fatty acid in energy production in light phase and increased the oxygen consumption during activities in dark phase. <b><i>Conclusions:</i></b> TRF effectively decreases hyperinsulinemia and restores pulsatile GH secretion in the overeating obese mice with significant improvement in substrate and energy metabolism and body composition without reducing total caloric intake.

<b><i>背景:</i></b> 代谢调控激素紊乱在肥胖进展过程中较为常见。关键调控激素轴——胰岛素-生长激素（GH）平衡，近来被提出可维持机体代谢谱特征。限时进食（TRF）是对抗肥胖的有效干预手段，但目前针对其脉冲式GH释放模式的相关研究仍较为匮乏。
<b><i>方法:</i></b> 本研究采用正常饲料喂养，在过度进食型黑皮质素4受体敲除（MC4RKO）肥胖小鼠模型中实施限时进食（TRF）干预。每日记录小鼠体重与食物摄入量。分别于TRF干预第5、8、9周采集系列血样，分别用于分析6小时脉冲式GH分泌谱、开展葡萄糖耐量试验及胰岛素耐量试验。于TRF干预第6周起，采用Phenomaster系统开展为期1周的间接量热检测；通过核磁共振波谱法（NMR）测定小鼠体成分。在实验终点采集肝脏与皮下白色脂肪组织，检测底物代谢与能量代谢相关基因的表达水平。
<b><i>结果:</i></b> 限时进食（TRF）可增强MC4RKO肥胖小鼠黑暗活动周期的脉冲式GH分泌，抑制高胰岛素血症，降低胰岛素/ GH比值。同时，该干预可改善胰岛素敏感性、代谢灵活性与葡萄糖耐量，减少血糖波动，并对底物代谢与脂肪组织褐变相关基因的表达进行适度调控。核磁共振波谱法（NMR）检测结果显示，TRF可降低小鼠体脂质量，增加瘦体重。间接量热检测结果表明，TRF可降低呼吸交换率（RER），提示其在光照周期中更多利用脂肪酸作为能量供能底物；同时可提升小鼠活动期（黑暗周期）的氧耗量。
<b><i>结论:</i></b> 综上，限时进食（TRF）可在不降低总热量摄入的前提下，有效改善过度进食型肥胖小鼠的高胰岛素血症，恢复其脉冲式GH分泌，同时显著改善底物与能量代谢及体成分组成。