BCKDK介导支链氨基酸代谢异常在恶病质骨骼肌蛋白合成的作用及机制

Ongoing skeletal muscle atrophy is the main characteristic of cancer cachexia and is correlated with the disease progression. The mechanism of muscle atrophy is still unknown and there is no effective therapeutic drug. Our previous study revealed that accelerated Branched Chain Amino Acid (BCAA) catabolism was associated with skeletal muscle atrophy during cancer cachexia. Branched α-Keto Acid Dehydrogenase (BCKD) Complex is the irreversible metabolic enzyme of BCAA catabolism. Dysfunction of BCKD Complex resulted in the skeletal muscle atrophy. BCKD Complex is regulated by BCKD kinase (BCKDK). Our preliminary experiment found that BCKDK expression was decreased in the murine model of muscle atrophy during cancer cachexia. The knockdown of BCKDK would result in the increase of BCKD Complex activity and the decrease of muscle protein synthesis. So we hypothesis that BCKDK inhibition mediated BCAA catabolic dysfunction contributes to the skeletal muscle atrophy. Activation of BCKDK is a new strategy for the prevention and treatment of cancer cachexia. To verify the hypothesis, we design in vitro and in vivo experiments to elucidate the following results. ①Change the expression and activity of BCKDK on the synthesis and degradation of muscle protein. ②Mechanism of BCKDK on the regulation of muscle protein synthesis. ③Effect of skeletal muscle tissue conditional knock-out or conditional knock-in BCKDK on muscle protein synthesis and degradation. ④Effect of BCKDK on the BCAA metabolism of serum and skeletal muscle tissue from the cachectic animal. The present study can not only reveal a novel mechanism of muscle atrophy during cachexia, but also provide potential drug targets for the prevention and treatment of cachexia.

进行性骨骼肌萎缩是肿瘤恶病质主要特征，且与疾病进展相关，机制尚未完全阐明，缺乏防治药物。我们前期研究发现支链氨基酸（BCAA）分解代谢加速与恶病质骨骼肌萎缩相关。支链α酮酸脱氢酶复合物（BCKDC）是BCAA代谢关键限制性酶，BCKDC功能异常升高会引起肌萎缩。BCKDC受其激酶BCKDK调控。近期预实验发现BCKDK在模型动物肌萎缩过程中表达下降，BCKDK可通过调控BCKDC活性而负向影响肌蛋白合成。为此假说：BCKDK抑制介导支链氨基酸代谢异常是骨骼肌萎缩新机制，激活BCKDK是肿瘤恶病质防治新策略。拟研究：①BCKDK表达和活性改变对肌蛋白合成/降解影响；②BCKDK调控肌蛋白合成的作用机制；③骨骼肌组织条件性敲除/敲入BCKDK对肌蛋白合成/降解影响；④BCKDK对模型动物血清和骨骼肌组织BCAA代谢调控作用。本项目有望阐明骨骼肌萎缩新机制，为肿瘤恶病质防治研究提供新思路/靶点。

肿瘤恶病质以骨骼肌萎缩为主要特征。骨骼肌萎缩取决于蛋白质的合成和降解速度。支链氨基酸BCAAs包括亮氨酸，异亮氨酸和缬氨酸，均是是哺乳动物的必需氨基酸，BCAAs直接在骨骼肌中分解代谢。BCKDK是调控骨骼肌蛋白合成的关键酶，我们研究发现：（1）Dex、TNFα及CM诱导C2C12萎缩的肌小管中均存在BCKDK蛋白低表达。（2）敲减BCKDK表达可抑制C2C12成肌细胞向C2C12肌小管的分化，而过表达BCKDK可促进C2C12成肌细胞分化。（3）敲减BCKDK可抑制肌蛋白合成，从而诱导C2C12肌小管萎缩，而过表达BCKDK促进肌蛋白合成，从而促进肌小管肥大。（4）特异性BCKDK抑制剂BT2可诱导C2C12肌小管萎缩，抑制肌蛋白合成促进肌蛋白降解。（5）过表达BCKDK可逆转Dex诱导的C2C12肌小管萎缩和肌蛋白合成下降。BCKDK敲减/过表达对C2C12肌小管中Akt/mTOR/FoxO3α信号通路中关键蛋白磷酸化有调控作用，是BCKDK调控骨骼肌萎缩的重要机制。（6）肿瘤恶病质小鼠骨骼肌中BCKDK mRNA和蛋白低表达。BCKDK敲减可导致小鼠腓肠肌质量减小、肌纤维萎缩、肌蛋白合成抑制、E3泛素连接酶表达上调。（7）BCKDK过表达可促进小鼠腓肠肌质量增加、肌纤维肥大、肌蛋白合成相关蛋白表达。（8）BCKDK过表达还可改善CT26肿瘤恶病质所致的骨骼肌萎缩和肌蛋白降解，促进肌蛋白合成。BCKDK敲减/过表达可调控小鼠腓肠肌Akt/mTOR信号通路，是其体内调控骨骼肌蛋白合成的主要机制之一。（9）BCKDK骨骼肌条件敲除动物荷瘤LLC肿瘤细胞后，动物生存期显著缩短，同时伴随骨骼肌萎缩和骨骼肌组织支链氨基酸代谢重编程。相关论文部分发表，并且部分成果在进一步研究。

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