在体外维持高功能的代谢上皮是具有挑战性的。随着时间的推移,原代人肝细胞(PHH)中的代谢受损主要是由于上皮-间质转化(EMT)。未成熟肝癌细胞系HepG2用作体外模型以探索增强肝表型的策略。表型表征包括测量尿素循环,脂质储存,三羧酸相关代谢物,活性氧,内质网钙外排,线粒体膜电位,耗氧率,和CYP450生物转化能力。用转录组学进行表达研究,免疫共沉淀和蛋白质组学。CRISPR/Cas9也用于基因工程化HepG2细胞。在确认PHHs发展为EMT表型后,发现tankyrase1/2的表达随时间增加。当阻断tankyrases1/2依赖性聚ADP核糖基化(PARylation)活性时,EMT恢复,通过生化和遗传扰动。Wnt/β-连环蛋白抑制剂XAV-939阻断tankyrase1/2,治疗提高了几个耗氧反应(电子传输链,OXHPOS,CYP450单氧化酶活性,I/II期异种生物生物转化,和餐时营业额),表明细胞代谢增强。在XAV-939条件下,谷胱甘肽依赖性氧化还原稳态也得到了显着改善。然后,在tankyrase1/2双敲除HepG2细胞中进行耗氧率和蛋白质组学实验,发现PARylation是有氧依赖性细胞呼吸的主要调节因子。此外,新的tankyrase1/2依赖性PARylation靶标,包括线粒体DLST,OGDH,被揭露。这项工作通过将PARylation与呼吸和新陈代谢联系起来,揭示了一个新的机制框架,从而扩大了这些重要过程背后的当前理解。XAV-939提出了一种直接和直接的策略来改善有氧活动,和新陈代谢,在(未成熟的)细胞培养物中。
The maintenance of a highly functional metabolic epithelium in vitro is challenging. Metabolic impairments in primary human hepatocytes (PHHs) over time is primarily due to epithelial-to-mesenchymal transitioning (EMT). The immature hepatoma cell line HepG2 was used as an in vitro model to explore strategies for enhancing the hepatic phenotype. The phenotypic characterization includes measuring the urea cycle, lipid storage, tricarboxylic acid-related metabolites, reactive oxygen species, endoplasmic reticulum calcium efflux, mitochondrial membrane potentials, oxygen consumptions rate, and CYP450 biotransformation capacity. Expression studies were performed with transcriptomics, co-immunoprecipitation and proteomics. CRISPR/Cas9 was also employed to genetically engineer HepG2 cells. After confirming that PHHs develop an EMT phenotype, expression of tankyrase1/2 was found to increase over time. EMT was reverted when blocking tankyrases1/2-dependent poly-ADP-ribosylation (PARylation) activity, by biochemical and genetic perturbation. Wnt/β-catenin inhibitor XAV-939 blocks tankyrase1/2 and treatment elevated several oxygen-consuming reactions (electron-transport chain, OXHPOS, CYP450 mono-oxidase activity, phase I/II xenobiotic biotransformation, and prandial turnover), suggesting that cell metabolism was enhanced. Glutathione-dependent redox homeostasis was also significantly improved in the XAV-939 condition. Oxygen consumption rate and proteomics experiments in tankyrase1/2 double knockout HepG2 cells then uncovered PARylation as master regulator of aerobic-dependent cell respiration. Furthermore, novel tankyrase1/2-dependent PARylation targets, including mitochondrial DLST, and OGDH, were revealed. This work exposed a new mechanistic framework by linking PARylation to respiration and metabolism, thereby broadening the current understanding that underlies these vital processes. XAV-939 poses an immediate and straightforward strategy to improve aerobic activities, and metabolism, in (immature) cell cultures.