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

<b><i>Introduction/Aim:</i></b> 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. <b><i>Methods:</i></b> 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 (<i>Bmal1</i> 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. <b><i>Results:</i></b> The steep profile, characterized by a higher HC exposure during <i>Bmal1</i>bathyphase, produced significant downregulation in 21 insulin sensitivity-related genes including <i>Insr, Irs1, Irs2, Pi3kca,</i> and <i>Adipor2</i>, compared to the flat and standard profile. Reduced intracellular IRS1 Tyr608, AKT Ser473, AMPK Thr172, and ACC Ser79 phosphorylations were also observed. <b><i>Conclusions:</i></b> 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：** 昼夜节律紊乱正逐渐成为代谢紊乱的危险因素，其中时钟基因的昼夜表达异常已被证实会影响胰岛素敏感性。近年来，昼夜节律系统与下丘脑-垂体-肾上腺（hypothalamus-pituitary-adrenal, HPA轴）之间的双向调控作用已得到广泛证实：昼夜节律可调控生理性内源性糖皮质激素（glucocorticoid, GC）的分泌与生理作用；反之，糖皮质激素亦是昼夜节律的强效调控因子，其不规范的给药方案与代谢损伤密切相关。本研究旨在通过体外实验，探讨不同浓度氢化可的松（hydrocortisone, HC）的每日暴露时序与骨骼肌胰岛素敏感性之间的相互作用。

**研究方法：** 将经血清休克同步化的小鼠骨骼肌C2C12细胞，分别暴露于三种模拟临床皮质醇分泌谱的氢化可的松环境：① 模拟健康人群生理性每日皮质醇分泌特征的标准皮质醇谱；② 接受每日一次缓释型氢化可的松治疗的肾上腺功能不全（adrenal insufficient, AI）患者的每日循环皮质醇谱（平坦皮质醇谱）；③ 接受每日三次常规速释型氢化可的松治疗的该类患者的每日循环皮质醇谱（陡峭皮质醇谱）。基于同步化C2C12细胞中时钟基因的24小时自发性振荡周期，将体外氢化可的松暴露的时间节点（即生物钟基因*Bmal1*的峰相、中相与谷相）与接受速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、下午6时）进行对齐。通过实时定量聚合酶链反应（RT-qPCR）与蛋白质印迹（Western blot）技术，分别检测84个胰岛素敏感性相关基因的转录水平及细胞内胰岛素信号蛋白的磷酸化水平。

**研究结果：** 相较于平坦谱与标准谱，陡峭皮质醇谱（其特征为在*Bmal1*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的磷酸化水平显著降低。

**研究结论：** 本研究证实，每日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与细胞内胰岛素信号通路。