Supplementary Material for: Irisin ameliorated skeletal muscle atrophy by inhibiting fatty acid oxidation and pyroptosis induced by palmitic acid in chronic kidney disease

Introduction:Protein-energy waste (PEW) is a common complication in patients with chronic kidney disease (CKD), among which skeletal muscle atrophy is one of the most important clinical features of PEW. Pyroptosis is a type of proinflammatory programmed cell death associated with skeletal muscle disease. Irisin, as a novel myokine, has attracted extensive attention for its protective role in the complications associated with CKD, but its role in muscle atrophy in CKD is unclear.Methods:Palmitic acid (PA) induced muscular atrophy was evaluated by a reduction in C2C12 myotube diameter. Muscle atrophy model was established in male C57BL/6J mice treated with 0.2% adenine for 4 weeks and then fed a 45% high-fat diet.BUN and Cr levels ,body and muscle weight, and muscle histology were assessed. The expression of carnitine palmitoyltransferase 1A (CPT1A) and pyroptosis-related protein was analysed by western blots or immunohistochemistry. The release of IL-1β was detected by ELISA.Results:In this study, we showed that PA induced muscular atrophy and manifested as a reduction in C2C12 myotube diameter. During this process PA can also induce pyroptosis, as shown by the upregulation of NLRP3, cleaved Caspase1 and GSDMD-N expression and the increased IL-1β release and PI-positive cell rate. Inhibition of Caspase1 or NLRP3 attenuated PA-induced pyroptosis and myotube atrophy in C2C12 cells. Importantly, Irisin treatment significantly ameliorated PA-induced skeletal muscle pyroptosis and atrophy. In terms of mechanism, PA upregulated CPT1A, a key enzyme of fatty acid oxidation(FAO), and Irisin attenuated this effect, which was consistent with Etomoxir (CPT1A inhibitor) treatment. Moreover, Irisin improved skeletal muscle atrophy and pyroptosis in adenine-induced mice by regulating FAO. Conclusion: our study firstly verifies that pyroptosis is a novel mechanism of skeletal muscle atrophy in CKD. Irisin ameliorated skeletal muscle atrophy by inhibiting FAO and pyroptosis in CKD, and Irisin may be developed as a potential therapeutic agent for the treatment of muscle wasting in CKD patients.

引言：蛋白质能量消耗（Protein-energy waste, PEW）是慢性肾脏病（chronic kidney disease, CKD）患者常见的并发症，其中骨骼肌萎缩是PEW最重要的临床特征之一。焦亡（pyroptosis）是一类与骨骼肌疾病相关的促炎程序性细胞死亡方式。鸢尾素（Irisin）作为一种新型肌因子，因其在CKD相关并发症中的保护作用受到广泛关注，但其在CKD相关性肌萎缩中的作用尚不清楚。
方法：通过检测C2C12肌管直径的变化，评估棕榈酸（Palmitic acid, PA）诱导的肌萎缩。构建雄性C57BL/6J小鼠肌萎缩模型：给予0.2%腺嘌呤喂养4周，随后饲喂45%高脂饮食。检测各组小鼠的血尿素氮（blood urea nitrogen, BUN）、肌酐（Cr）水平、体质量与肌质量，以及肌肉组织病理学特征。采用蛋白质印迹法或免疫组织化学法检测肉碱棕榈酰转移酶1A（carnitine palmitoyltransferase 1A, CPT1A）及焦亡相关蛋白的表达水平。通过酶联免疫吸附试验（ELISA）检测IL-1β的释放量。
结果：本研究证实，PA可诱导C2C12肌管直径降低，从而构建肌萎缩模型；在此过程中，PA还可诱导焦亡发生，具体表现为NLRP3、活化半胱氨酸天冬氨酸蛋白酶-1（cleaved Caspase1）及GSDMD-N的表达上调，同时IL-1β释放量增加、碘化丙啶（PI）阳性细胞比例升高。抑制Caspase1或NLRP3的活性，可减轻PA诱导的C2C12细胞焦亡及肌管萎缩。重要的是，鸢尾素处理可显著改善PA诱导的骨骼肌焦亡与肌萎缩。机制层面，PA可上调脂肪酸氧化（fatty acid oxidation, FAO）的关键酶CPT1A的表达，而鸢尾素可抵消这一效应，该结果与依托莫昔（Etomoxir，CPT1A抑制剂）的处理效果一致。此外，鸢尾素可通过调控FAO改善腺嘌呤诱导的小鼠骨骼肌萎缩与焦亡。
结论：本研究首次证实，焦亡是CKD相关性骨骼肌萎缩的全新发病机制。鸢尾素可通过抑制FAO与焦亡，改善CKD患者的骨骼肌萎缩，有望成为治疗CKD患者肌消耗的潜在靶向治疗药物。