Lim Pooreum, Woo Sang Woo, Han Jihye, Lee Young Lim, Lim Jin Ju, Kang Yeong Hoon, Moon Ji Wook, Nam Jeong Min, Kim Jeong Hyeon, Kim Donghun, Shim Jae Ho, Kim Hyeon Soo
Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Korea.
J Cachexia Sarcopenia Muscle. 2025 Aug;16(4):e70044. doi: 10.1002/jcsm.70044.
Cancer-associated cachexia (CAC) is a multifactorial syndrome characterised by progressive loss of muscle mass with limited Food and Drug Administration treatments. Although emerging evidence suggests that l-leucine and β-hydroxy-β-methyl butyrate (HMB) have potential for treating CAC, the role of α-ketoisocaproate (KIC), a metabolite of l-leucine, remains unclear. Therefore, this study explored the use of KIC as a therapeutic agent for CAC-induced muscle atrophy by targeting myostatin.
We evaluated the effect of KIC on muscle atrophy using BALB/c mice and C2C12 myotubes as models of C26- and 4T1-induced CAC. Male and female mice were injected with C26 and 4T1 cells, respectively. Grip strength was measured weekly, and mice were sacrificed 4 weeks post-injection for muscle collection. C2C12 myotubes were treated with conditioned media (CM) derived from C26 or 4T1 cells.
KIC suppressed mRNA expression of myostatin, a key regulator of muscle atrophy, more effectively than did l-leucine (-26.37 ± 4.11%, p < 0.01). KIC enhanced protein turnover in C2C12 myotubes and maintained 50% cell viability at high concentrations (KIC: 4.68 mM, HMB: 3.11 mM). Following CM treatment, KIC suppressed MuRF1 and MAFbx expression in a myostatin-dependent manner, thereby reducing their polyubiquitination. KIC restored Akt-FoxO3a phosphorylation, leading to improved myotube diameter (+63.8 ± 25.71%, p < 0.05) and fusion index (+51.9 ± 22.6%, p < 0.05). Immunofluorescence and nuclear fractionation revealed that KIC reduced FoxO3a nuclear accumulation. CM reduced p-Akt-FoxO3a interaction, which was rescued by KIC. In vivo, KIC administration increased body weight (11.11 ± 8.53%), grip strength (24.76 ± 10.58%), and skeletal muscle mass (p < 0.001) in C26 tumour-bearing mice. Protein expression of myostatin in the tibialis anterior (TA) muscle (-23.57 ± 12.22%, p < 0.05) and serum (-52.11 ± 3.56%, p < 0.001) was lower in KIC-treated mice (n = 12) compared with that in the controls. KIC increased the mean fibre cross-sectional area in TA (24.51 ± 14.14%, p < 0.01). In 4T1 tumour-bearing mice, KIC improved body weight (13.10 ± 10.76%) and grip strength (7.42 ± 4.33%) (p < 0.001, n = 10). Serum myostatin levels (-57.43 ± 9.46%, p < 0.001) and skeletal muscle weight were reduced in KIC-treated mice (n = 10).
Our findings demonstrate that KIC improves muscle function in CAC-induced muscle atrophy by regulating myostatin expression in skeletal muscle via the Akt-FoxO3a pathway. Thus, KIC has been proposed as a potential therapeutic agent against CAC.
癌症相关性恶病质（CAC）是一种多因素综合征，其特征为肌肉质量逐渐丧失，而美国食品药品监督管理局批准的治疗方法有限。尽管新出现的证据表明L-亮氨酸和β-羟基-β-甲基丁酸酯（HMB）具有治疗CAC的潜力，但L-亮氨酸的代谢产物α-酮异己酸（KIC）的作用仍不清楚。因此，本研究通过靶向肌肉生长抑制素，探索了KIC作为治疗CAC诱导的肌肉萎缩的治疗剂的用途。
我们使用BALB/c小鼠和C2C12肌管作为C26和4T1诱导的CAC模型，评估了KIC对肌肉萎缩的影响。分别给雄性和雌性小鼠注射C26和4T1细胞。每周测量握力，注射后4周处死小鼠以收集肌肉。用来自C26或4T1细胞的条件培养基（CM）处理C2C12肌管。
与L-亮氨酸相比，KIC更有效地抑制了肌肉萎缩关键调节因子肌肉生长抑制素的mRNA表达（-26.37±4.11%，p<0.01）。KIC增强了C2C12肌管中的蛋白质周转，并在高浓度下维持50%的细胞活力（KIC：4.68 mM，HMB：3.11 mM）。CM处理后，KIC以肌肉生长抑制素依赖性方式抑制MuRF1和MAFbx表达，从而减少它们泛素化。KIC恢复了Akt-FoxO3a磷酸化，导致肌管直径增加（+63.8±25.71%，p<0.05）和融合指数增加（+51.9±22.6%，p<0.05）。免疫荧光和细胞核分级分离显示，KIC减少了FoxO3a的核积累。CM降低了p-Akt-FoxO3a相互作用，而KIC可使其恢复。在体内，给予KIC可增加C26荷瘤小鼠的体重（11.11±8.53%）、握力（24.76±10.58%）和骨骼肌质量（p<0.001）。与对照组相比，KIC处理组小鼠（n=12）胫前肌（TA）中肌肉生长抑制素的蛋白表达（-23.57±12.22%，p<0.05）和血清中肌肉生长抑制素的蛋白表达（-52.11±3.56%，p<0.001）较低。KIC增加了TA的平均纤维横截面积（24.51±14.14%，p<0.01）。在4T1荷瘤小鼠中，KIC改善了体重（13.10±10.76%）和握力（7.42±4.33%）（p<0.001，n=10）。KIC处理组小鼠（n=10）的血清肌肉生长抑制素水平（-57.43±9.46%，p<0.001）和骨骼肌重量降低。
我们的研究结果表明，KIC通过Akt-FoxO3a途径调节骨骼肌中肌肉生长抑制素的表达，从而改善CAC诱导的肌肉萎缩中的肌肉功能。因此，KIC被认为是一种潜在的抗CAC治疗剂。
