Supplementary Material for: Cortisol Circadian Rhythm and Insulin Resistance in Muscle: Effect of Dosing and Timing of Hydrocortisone Exposure on Insulin Sensitivity in Synchronized Muscle Cells

Introduction/Aim: Circadian clock disruption is emerging as a risk factor for metabolic disorders, and particularly, alterations in clock genes circadian expression have been shown to influence insulin sensitivity. Recently, the reciprocal interplay between the circadian clock machinery and hypothalamus-pituitary-adrenal axis has been largely demonstrated: the circadian clock may control the physiological circadian endogenous glucocorticoid (GC) secretion and action; GCs, in turn, are potent regulators of the circadian clock and their inappropriate replacement has been associated with metabolic impairment. The aim of the current study was to investigate in vitro the interaction between the timing-of-the-day exposure to different hydrocortisone (HC) concentrations and muscle insulin sensitivity. Methods: Serum-shock synchronized mouse skeletal muscle C2C12 cells were exposed to different HC concentrations resembling the circulating daily physiological cortisol profile (standard cortisol profile) and the circulating daily cortisol profile that reached in adrenal insufficient (AI) patients treated with once-daily modified-release HC (flat cortisol profile) and treated with thrice-daily conventional immediate-release HC (steep cortisol profile). The 24 h spontaneous oscillation of the clock genes in synchronized C2C12 cells was used to align the timing for in vitro HC exposure (Bmal1 acrophase, midphase, and bathyphase) with the reference times of cortisol peaks in AI patients treated with IR-HC (8 a.m., 1 p.m., and 6 p.m.). A panel of 84 insulin sensitivity-related genes and intracellular insulin signaling proteins were analyzed by RT-qPCR and Western blot, respectively. Results: The steep profile, characterized by a higher HC exposure during Bmal1bathyphase, produced significant downregulation in 21 insulin sensitivity-related genes including Insr, Irs1, Irs2, Pi3kca, and Adipor2, compared to the flat and standard profile. Reduced intracellular IRS1 Tyr608, AKT Ser473, AMPK Thr172, and ACC Ser79 phosphorylations were also observed. Conclusions: The current study demonstrated that late-in-the-day cortisol exposure modulates insulin sensitivity-related gene expression and intracellular insulin signaling in skeletal muscle cells.

研究背景与目的：昼夜节律紊乱现已成为代谢紊乱的潜在危险因素，其中时钟基因的昼夜表达异常已被证实可影响胰岛素敏感性。近期学界已广泛证实，昼夜节律系统与下丘脑-垂体-肾上腺轴（hypothalamus-pituitary-adrenal axis）之间存在双向交互调控：昼夜节律可调控内源性糖皮质激素（glucocorticoid, GC）的生理性昼夜分泌与作用；反之，糖皮质激素亦是调控昼夜节律的强效因子，其不当补充与代谢损伤密切相关。本研究旨在体外探讨不同氢化可的松（hydrocortisone, HC）浓度的日间暴露时机与肌肉胰岛素敏感性之间的相互作用。
方法：采用血清休克同步化的小鼠骨骼肌C2C12细胞，分别暴露于模拟三种临床场景的氢化可的松浓度梯度：其一为模拟健康个体的生理性每日皮质醇分泌谱（标准皮质醇谱）；其二为接受每日一次缓释氢化可的松治疗的肾上腺功能不全（adrenal insufficient, AI）患者的每日皮质醇分泌谱（平稳皮质醇谱）；其三为接受每日三次常规即释型氢化可的松治疗的肾上腺功能不全患者的每日皮质醇分泌谱（峰状皮质醇谱）。本研究以同步化C2C12细胞中时钟基因的24小时自发性振荡为参照，将体外氢化可的松暴露的时机（即Bmal1基因的峰相、中相及谷相）与接受即释型氢化可的松治疗的肾上腺功能不全患者的皮质醇峰值参考时间（上午8时、下午1时及下午6时）进行对齐。分别通过实时荧光定量聚合酶链反应（RT-qPCR）与蛋白质印迹法（Western blot），检测84个胰岛素敏感性相关基因及细胞内胰岛素信号蛋白的表达水平。
结果：相较于平稳皮质醇谱与标准皮质醇谱，峰状皮质醇谱（其特征为在Bmal1基因谷相阶段暴露更高浓度的氢化可的松）可使包括Insr、Irs1、Irs2、Pi3kca及Adipor2在内的21个胰岛素敏感性相关基因出现显著下调。同时还观察到细胞内IRS1 Tyr608、AKT Ser473、AMPK Thr172及ACC Ser79的磷酸化水平降低。
结论：本研究证实，日间后期的皮质醇暴露可调控骨骼肌细胞内胰岛素敏感性相关基因的表达及细胞内胰岛素信号通路。