BackgroundSkeletal muscle is the primary organ involved in insulin‐mediated glucose metabolism. Elevated levels of CILP2 are a significant indicator of impaired glucose tolerance and are predominantly expressed in skeletal muscle. It remains unclear whether CILP2 contributes to age‐related muscle atrophy through regulating the glucose homeostasis and insulin sensitivity.MethodsInitially, the expression levels of CILP2 were assessed in elderly mice and patients with sarcopenia. Lentiviral vectors were used to induce either silencing or overexpression of CILP2 in C2C12 myoblast cells. The effects of CILP2 on proliferation, myogenic differentiation, insulin sensitivity and glucose uptake were evaluated using immunofluorescence, western blotting, real‐time quantitative polymerase chain reaction, RNA sequencing, glucose uptake experiments, dual‐luciferase reporter assays and co‐immunoprecipitation (CO‐IP). An adeno‐associated virus‐9 containing a muscle‐specific promoter was injected into SAMP8 senile mice to observe the efficacy of CILP2 knockout.ResultsWe found that there was more CLIP2 expressed in the skeletal muscle of ageing mice (+1.1‐fold, p < 0.01) and in patients with sarcopenia (+2.5‐fold, p < 0.01) compared to the control group. Following the overexpression of CILP2, Ki67 (−65%, p < 0.01), PCNA (−32%, p < 0.05), MyoD1 (−89%, p < 0.001), MyoG (−31%, p < 0.05) and MyHC (−85%, p < 0.001), which indicate proliferation and differentiation potential, were significantly reduced. In contrast, MuRF‐1 (+59%, p < 0.05), atrogin‐1 (+43%, p < 0.05) and myostatin (+31%, p < 0.05), the markers of muscular atrophy, were significantly increased. Overexpression of CILP2 decreased insulin sensitivity, glucose uptake (−18%, p < 0.001), GLUT4 translocation to the membrane and the maximum respiratory capacity of mitochondria. Canonical Wnt signalling was identified through RNA sequencing as a potential pathway for CILP2 regulation in C2C12, and Wnt3a was confirmed as an interacting protein of CILP2 in the CO‐IP assay. The addition of recombinant Wnt3a protein reversed the inhibitory effects on myogenesis and glucose metabolism caused by CILP2 overexpression. Conversely, CILP2 knockdown promoted myogenesis and glucose metabolism. CILP2 knockdown improved muscle atrophy in mice, characterized by significant increases in time to exhaustion (+42%, p < 0.001), grip strength (+19%, p < 0.01), muscle mass (+15%, p < 0.001) and mean muscle cross‐sectional area (+37%, p < 0.01). CILP2 knockdown enhanced glycogen synthesis (+83%, p < 0.001) and the regeneration of oxidative and glycolytic muscle fibres in SAMP8 ageing mice via the Wnt/β‐catenin signalling pathway.ConclusionsOur results indicate that CILP2 interacts with Wnt3a to suppress the Wnt/β‐catenin signalling pathway and its downstream cascade, leading to impaired insulin sensitivity and glucose metabolism in skeletal muscle. Targeting CILP2 inhibition could offer potential therapeutic benefits for sarcopenia.

中文翻译：

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抑制 CILP2 通过 Wnt 信号通路改善肌肉减少症的葡萄糖代谢和线粒体功能障碍


背景骨骼肌是参与胰岛素介导的葡萄糖代谢的主要器官。CILP2 水平升高是葡萄糖耐量受损的重要指标，主要在骨骼肌中表达。目前尚不清楚 CILP2 是否通过调节葡萄糖稳态和胰岛素敏感性导致与年龄相关的肌肉萎缩。方法最初，评估老年小鼠和肌肉减少症患者 CILP2 的表达水平。慢病毒载体用于诱导 C2C12 成肌细胞中 CILP2 的沉默或过表达。使用免疫荧光、蛋白质印迹、实时定量聚合酶链反应、RNA 测序、葡萄糖摄取实验、双荧光素酶报告基因测定和免疫共沉淀 （CO-IP） 评估 CILP2 对增殖、肌源性分化、胰岛素敏感性和葡萄糖摄取的影响。将含有肌肉特异性启动子的腺相关病毒 9 注射到 SAMP8 老年小鼠中，以观察 CILP2 敲除的疗效。结果我们发现，与对照组相比，衰老小鼠骨骼肌中 CLIP2 的表达更多（+1.1 倍，p < 0.01）和肌肉减少症患者（+2.5 倍，p < 0.01）。过表达 CILP2 后，Ki67 （-65%，p < 0.01）、PCNA （-32%，p < 0.05）、MyoD1 （-89%，p < 0.001）、MyoG （-31%，p < 0.05） 和 MyHC （-85%，p < 0.001），表明增殖和分化潜力，显着降低。相比之下，肌肉萎缩的标志物 MuRF-1 （+59%，p < 0.05）、atrogin-1 （+43%，p < 0.05） 和肌肉生长抑制素 （+31%，p < 0.05） 显著增加。CILP2 的过表达降低了胰岛素敏感性、葡萄糖摄取 （-18%，p < 0.001）、GLUT4 易位到膜和线粒体的最大呼吸能力。通过 RNA 测序确定经典 Wnt 信号转导是 C2C12 中 CILP2 调节的潜在途径，Wnt3a 在 CO-IP 测定中被证实为 CILP2 的相互作用蛋白。重组 Wnt3a 蛋白的添加逆转了 CILP2 过表达对肌生成和葡萄糖代谢的抑制作用。相反，CILP2 敲低促进肌生成和葡萄糖代谢。CILP2 敲低改善了小鼠的肌肉萎缩，其特征是筋疲力尽时间显著增加 （+42%，p < 0.001）、握力 （+19%，p < 0.01）、肌肉质量 （+15%，p < 0.001） 和平均肌肉横截面积 （+37%，p < 0.01）。CILP2 敲低通过 Wnt/β-catenin 信号通路增强了 SAMP8 衰老小鼠的糖原合成 （+83%，p < 0.001） 以及氧化和糖酵解肌纤维的再生。结论我们的结果表明，CILP2 与 Wnt3a 相互作用，抑制 Wnt/β-catenin 信号通路及其下游级联反应，导致骨骼肌胰岛素敏感性和葡萄糖代谢受损。靶向 CILP2 抑制可能为肌肉减少症提供潜在的治疗益处。