Metabolite Profiles Reveal Energy Failure and Impaired Beta-Oxidation in Liver of Mice with Complex III Deficiency Due to a BCS1L Mutation

Background & AimsLiver is a target organ in many mitochondrial disorders, especially if the complex III assembly factor BCS1L is mutated. To reveal disease mechanism due to such mutations, we have produced a transgenic mouse model with c.232A>G mutation in Bcs1l, the causative mutation for GRACILE syndrome. The homozygous mice develop mitochondrial hepatopathy with steatosis and fibrosis after weaning. Our aim was to assess cellular mechanisms for disease onset and progression using metabolomics. MethodsWith mass spectrometry we analyzed metabolite patterns in liver samples obtained from homozygotes and littermate controls of three ages. As oxidative stress might be a mechanism for mitochondrial hepatopathy, we also assessed H2O2 production and expression of antioxidants. ResultsHomozygotes had a similar metabolic profile at 14 days of age as controls, with the exception of slightly decreased AMP. At 24 days, when hepatocytes display first histopathological signs, increases in succinate, fumarate and AMP were found associated with impaired glucose turnover and beta-oxidation. At end stage disease after 30 days, these changes were pronounced with decreased carbohydrates, high levels of acylcarnitines and amino acids, and elevated biogenic amines, especially putrescine. Signs of oxidative stress were present in end-stage disease. ConclusionsThe findings suggest an early Krebs cycle defect with increases of its intermediates, which might play a role in disease onset. During disease progression, carbohydrate and fatty acid metabolism deteriorate leading to a starvation-like condition. The mouse model is valuable for further investigations on mechanisms in mitochondrial hepatopathy and for interventions.

背景与目的
肝脏是多种线粒体疾病的靶器官，当复合物III组装因子BCS1L发生突变时尤为如此。为阐明此类突变所致的疾病机制，我们构建了携带Bcs1l基因c.232A>G突变的转基因小鼠模型，该突变为GRACILE综合征（GRACILE syndrome）的致病突变。纯合子小鼠在断奶后会出现伴有脂肪变性与纤维化的线粒体肝病。本研究旨在通过代谢组学（metabolomics）分析，评估疾病发生与进展的细胞机制。

方法
我们采用质谱技术（mass spectrometry）分析了三个年龄组的纯合子小鼠及其同窝对照小鼠的肝脏样本代谢谱。鉴于氧化应激（oxidative stress）可能是线粒体肝病的发病机制之一，我们同时检测了过氧化氢（H₂O₂）生成量与抗氧化相关分子的表达水平。

结果
14日龄时，纯合子小鼠的代谢谱与对照组相似，仅腺苷一磷酸（AMP）水平略有降低。24日龄时，肝细胞已出现首批组织病理学改变，此时琥珀酸、延胡索酸与AMP水平升高，与葡萄糖周转受损及β-氧化（beta-oxidation）异常相关。30日龄进入疾病终末期时，上述代谢改变更为显著：碳水化合物水平降低，酰基肉碱（acylcarnitines）与氨基酸水平升高，生物源性胺（biogenic amines，尤其是腐胺putrescine）水平上升。终末期疾病可见明确的氧化应激相关指标异常。

结论
本研究结果提示，早期三羧酸循环（Krebs cycle）缺陷伴中间产物蓄积，可能在疾病发病中发挥关键作用。疾病进展过程中，碳水化合物与脂肪酸代谢受损，最终导致类饥饿状态。该小鼠模型可为线粒体肝病的发病机制研究及干预策略开发提供宝贵的实验工具。