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-能量浪费（PEW）是慢性肾脏病（CKD）患者常见的并发症之一，其中骨骼肌萎缩是PEW最重要的临床特征之一。焦亡是一种与骨骼肌疾病相关的促炎症性程序性细胞死亡。作为新型肌细胞因子，Irisin因其对CKD相关并发症的保护作用而受到广泛关注，但其对CKD中肌肉萎缩的作用尚不明确。方法：通过C2C12肌管直径的减小来评估棕榈酸（PA）诱导的肌肉萎缩。在用0.2%腺嘌呤处理的雄性C57BL/6J小鼠中建立肌肉萎缩模型，持续4周，随后喂以45%高脂肪饮食。评估尿素氮（BUN）和肌酐（Cr）水平、体重和肌肉重量，以及肌肉组织学。通过Western印迹或免疫组化分析肉碱棕榈酰转移酶1A（CPT1A）和焦亡相关蛋白的表达。通过ELISA检测IL-1β的释放。结果：在本研究中，我们证明了PA诱导的肌肉萎缩表现为C2C12肌管直径的减小。在此过程中，PA还可以诱导焦亡，如NLRP3、裂解的Caspase1和GSDMD-N表达的上调以及IL-1β释放和PI阳性细胞率的增加所示。抑制Caspase1或NLRP3可减轻PA诱导的焦亡和C2C12细胞的肌管萎缩。重要的是，Irisin治疗显著改善了PA诱导的骨骼肌焦亡和萎缩。在机制方面，PA上调了脂肪酸氧化（FAO）的关键酶CPT1A，而Irisin减弱了这一作用，这与Etomoxir（CPT1A抑制剂）治疗一致。此外，Irisin通过调节FAO改善了腺嘌呤诱导的小鼠的骨骼肌萎缩和焦亡。结论：我们的研究首次证实了焦亡是CKD中骨骼肌萎缩的新机制。Irisin通过抑制CKD中的FAO和焦亡来改善骨骼肌萎缩，Irisin可能被开发为治疗CKD患者肌肉消耗的潜在治疗剂。