Improved oxygenation dramatically alters metabolism and gene expression in cultured primary mouse hepatocytes.
Improved oxygenation dramatically alters metabolism and gene expression in cultured primary mouse hepatocytes.
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DOI：
10.1002/hep4.1140
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发表时间：
2018-03
影响因子：
5.1
通讯作者：
Oude Elferink RPJ
中科院分区：
文献类型：
--
作者：
Gilglioni EH;Chang JC;Duijst S;Go S;Adam AAA;Hoekstra R;Verhoeven AJ;Ishii-Iwamoto EL;Oude Elferink RPJ
原代肝细胞培养是研究肝功能的重要体外系统。在体内，肝细胞具有高氧化代谢。然而，在体外静态培养中，通过扩散方式的氧气供应远低于体内血液循环。因此，我们研究了缺氧是否有助于培养肝细胞的去分化和代谢失调。为此，将鼠肝细胞在胶原夹层中在静态或振荡（60转/分钟）条件下培养。通过培养基和细胞中的代谢物、缺氧诱导因子（HIF）-1/2 α蛋白质印迹法以及缺氧诱导因子靶基因和糖脂代谢关键基因的真实的实时定量聚合酶链反应来检测缺氧对肝细胞培养物的影响。与静态培养相比，振摇培养中的肝细胞显示出较低的糖酵解活性和甘油三酯积累，与改善的氧输送和线粒体能量代谢相一致。一致的是，静态培养物显示出显著的HIF-2α表达，这在新鲜分离的肝细胞和振摇培养物中检测不到。HIF靶基因的转录水平与静态培养物相比，振荡培养物中甘油醛3磷酸脱氢酶[Gapdh]、葡萄糖转运蛋白1 [Glut 1]、丙酮酸脱氢酶激酶1 [Pdk 1]和乳酸脱氢酶A [Ldha]以及脂质代谢关键基因，如肉毒碱棕榈酰转移酶1（Cpt 1）、载脂蛋白B（Apo B）和乙酰辅酶A羧化酶1（Acc 1）的表达显著降低。此外，由于氧气输送的改善，肝细胞核因子4α（Hnf 4 α）和法尼醇X受体（Fxr）的表达在振荡培养物中得到了更好的保留。我们进一步揭示了HIF-2信号转导参与了缺氧诱导的Fxr下调。结论：小鼠原代肝细胞静态培养存在缺氧现象。通过简单的摇动改善氧合防止代谢酶表达的重大变化和异常甘油三酯积累;此外，它更好地维持细胞的分化状态。因此，振摇培养是使用原代肝细胞作为体外模型的可取策略。（Hepatology Communications 2018;2：299 - 312）
Primary hepatocyte culture is an important in vitro system for the study of liver functions. In vivo, hepatocytes have high oxidative metabolism. However, oxygen supply by means of diffusion in in vitro static cultures is much less than that by blood circulation in vivo. Therefore, we investigated whether hypoxia contributes to dedifferentiation and deregulated metabolism in cultured hepatocytes. To this end, murine hepatocytes were cultured under static or shaken (60 revolutions per minute) conditions in a collagen sandwich. The effect of hypoxia on hepatocyte cultures was examined by metabolites in media and cells, hypoxia‐inducible factors (HIF)‐1/2α western blotting, and real‐time quantitative polymerase chain reaction for HIF target genes and key genes of glucose and lipid metabolism. Hepatocytes in shaken cultures showed lower glycolytic activity and triglyceride accumulation than static cultures, compatible with improved oxygen delivery and mitochondrial energy metabolism. Consistently, static cultures displayed significant HIF‐2α expression, which was undetectable in freshly isolated hepatocytes and shaken cultures. Transcript levels of HIF target genes (glyceraldehyde 3‐phosphate dehydrogenase [Gapdh], glucose transporter 1 [Glut1], pyruvate dehydrogenase kinase 1 [Pdk1], and lactate dehydrogenase A [Ldha]) and key genes of lipid metabolism, such as carnitine palmitoyltransferase 1 (Cpt1), apolipoprotein B (Apob), and acetyl‐coenzyme A carboxylase 1 (Acc1), were significantly lower in shaken compared to static cultures. Moreover, expression of hepatocyte nuclear factor 4α (Hnf4α) and farnesoid X receptor (Fxr) were better preserved in shaken cultures as a result of improved oxygen delivery. We further revealed that HIF‐2 signaling was involved in hypoxia‐induced down‐regulation of Fxr. Conclusion: Primary murine hepatocytes in static culture suffer from hypoxia. Improving oxygenation by simple shaking prevents major changes in expression of metabolic enzymes and aberrant triglyceride accumulation; in addition, it better maintains the differentiation state of the cells. The shaken culture is, therefore, an advisable strategy for the use of primary hepatocytes as an in vitro model. (Hepatology Communications 2018;2:299‐312)
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DOI：
10.1007/978-1-59745-019-5_13
发表时间：
2010-01-01
期刊：
MOUSE CELL CULTURE: METHODS AND PROTOCOLS
影响因子：
--
作者：
Li, Wan-Chun;Ralphs, Kate L.;Tosh, David
通讯作者：
Tosh, David
影响因子：
14.9
作者：
Benita Y;Kikuchi H;Smith AD;Zhang MQ;Chung DC;Xavier RJ
通讯作者：
Xavier RJ
影响因子：
56.9
作者：
Jaakkola, P;Mole, DR;Ratcliffe, PJ
通讯作者：
Ratcliffe, PJ
影响因子：
64.8
作者：
Maxwell, PH;Wiesener, MS;Ratcliffe, PJ
通讯作者：
Ratcliffe, PJ
影响因子：
2.9
作者：
BLAKE, DA;MCLEAN, NV
通讯作者：
MCLEAN, NV