应用替吡法尼阻断瓦伯格效应对抗烧伤后胰岛素抵抗

Metabolic derangements, including hypermetabolism, insulin resistance, hyperlactatemia, and muscle wasting, are major complications of burn injury and significantly affect clinical outcome. The predominant assumption has been that insulin resistance plays a pivotal role in these metabolic aberrations. However, the currently prescribed insulin-sensitizers have limited utility in burn patients owing to their adverse side effects. Despite decades of research, no new treatments have emerged to target stress-induced insulin resistance or metabolic dysfunction. Hence, new strategies need to be developed. A recent investigation to identify new targets showed that burn injury increases protein farnesylation and that farnesyltransfrase inhibitor (FTI) reverses burn-induced muscle insulin resistance, but the underlying molecular mechanisms remains unclear. The Warburg effect was originally described as the metabolic reprogramming of cancer cells such that glycolytic ATP synthesis predominates over mitochondrial oxidative phosphorylation even under normoxia. Recently, the Warburg effect was recognized as a component of the inflammatory response associated with various inflammatory diseases, including critical illness. Preliminary results showed that burn induced the Warburg effect in mouse muscle as indicated by the induction of hypoxia-inducible factor-1a (HIF-1a), a hallmark of the Warburg effect, and by increased aerobic glycolysis under normoxic conditions, all of which were reversed by FTI. Additionally, the data in cultured muscle cells indicate that farnesylation of LKB1 (a master kinase in the regulation of metabolism) mediates cytokine-induced HIF-1a expression and thereby induces insulin resistance. It is hypothesized that burn induces the Warburg effect which in turn causes and/or exacerbates insulin resistance in skeletal muscle. This project is designed to determine whether the Warburg effect is an appropriate target for reversing burn-induced insulin resistance and metabolic disturbance. This proposal is expected to provide scientific rationale to develop a clinical trial of FTIs in burn patients.

严重烧伤诱发的胰岛素抵抗危害大，难以纠正，胰岛素增敏剂不适用于烧伤患者，为探索新的治疗策略，我们着眼于瓦伯格效应。瓦伯格效应是指葡萄糖的无氧酵解代替线粒体的氧化磷酸化，成为细胞的主要供能方式，其广泛存在于包括严重烧伤在内的多种炎性疾病中。烧伤诱发的瓦伯格效应不是对缺氧的暂时适应，而是基因诱导下的代谢重组。而这种代谢重组中，缺氧诱导因子HIF-1α通路很可能会被激活，并通过肝激酶B1法尼基化导致或加剧了烧伤后肌肉的胰岛素抵抗。据此我们提出假说：瓦伯格效应通过肝激酶B1法尼基化导致或加剧了烧伤后肌肉的胰岛素抵抗，法尼酰基转移酶抑制剂FTI治疗可以逆转烧伤和细胞因子诱发的HIF-1α在小鼠肌肉和C2C12肌管中的表达，从而达到对抗烧伤后胰岛素抵抗的作用。我们将尝试证明这一假说，从而确定瓦伯格效应是对抗烧伤诱发的胰岛素抵抗的适当目标，为烧伤患者临床应用FTI-替吡法尼的临床试验提供科学依据。

严重烧伤会导致胰岛素抵抗并伴随瓦伯格效应。瓦伯格效应的特征为糖酵解产生ATP提供的能量，取代线粒体的氧化磷酸化作用，这种代谢方式产能效率低，但速率却更高。严重烧伤的突出特点为代谢紊乱，这包括高血糖、胰岛素抵抗、负担平衡等。目前的观点认为，胰岛素抵抗是主要的诱发因素之一。而近年来，部分学者针对严重烧伤后的瓦伯格效应与法尼基化蛋白与胰岛素抵抗的关系进行了研究探索。本研究以25-30％全身体表面积(TBSA)深Ⅱ-Ⅲ°烫伤小鼠为模型，基于模型基础上，探讨法尼酰基转移酶抑制剂替吡法尼能否影响小鼠烧伤诱发的肌肉胰岛素抵抗。研究结果显示：烫伤后第3天， HIF-1α、PKM2显著表达，第1、3、7天烫伤治疗组HOMA-IR值均低于烫伤对照组，但只在伤后1天、3天均值比较差异有统计学意义(p<0.05)。各个时间点烫伤治疗组HIF-1α/ GAPDH值均低于烫伤对照组，伤后3天均值比较差异有统计学意义(p<0.05)。另外，为进一步推测替吡法尼针对烧伤后胰岛素抵抗可能的作用途径。我们构建了肌肉特异性PKM2敲除（-/-）（M-PKM2-KO）和肌肉特异性FTase-/-（M-FTase-KO）敲除，LKB1C431S敲入型C57BL/6小鼠。将内源性LKB1被敲除后，重新引入到C2C12细胞中野生型和抗法尼基化的LKB1突变体（LKB1C433S）。而LKB1C344S相较于野生型LKB1，能够减弱细胞因子诱导的HIF-1α表达，从而提示LKB1法尼基化在细胞因子诱导的HIF-1α表达起着重要作用。