Institute of Anatomy Medical Faculty, Otto-Von-Guericke University, 39120, Magdeburg, Germany.
Leibniz Institute for Neurobiology, Otto-Von-Guericke University, 39120, Magdeburg, Germany.
Histochem Cell Biol. 2023 May;159(5):389-400. doi: 10.1007/s00418-023-02180-x. Epub 2023 Feb 15.
An improved oxygen availability in air-liquid interface (ALI) cultures of enterocytes of the small intestine seems to be primarily responsible for morphological, metabolic, and functional changes. Intestinal porcine epithelial cells 1 (IPEC-1) are less investigated and are rarely used as model for intestinal barrier but showed a profound change of cell shape during ALI cultivation. We aim to answer the following question: Are the observed morphological effects accompanied by changes in metabolic function? A microarray analysis of submerged culture (SMC) and ALI cultures identified 830 significantly regulated genes. Subsequent functional clustering revealed alterations in 31 pathways, with the highest number of regulated genes in metabolic pathways, carbon metabolism, glycolysis, and hypoxia-inducible factor (HIF) signaling. Furthermore, HIF-1α as a mediator of a metabolic switch between glycolysis and oxidative phosphorylation showed a trend of increased mRNA levels in ALI in contrast to a reduced nuclear HIF-1α content in the nucleus. Candidate genes of oxidative phosphorylation such as a mitochondrial marker exhibited enhanced mRNA levels, which was confirmed by western blot analysis. Cytochrome C oxidase (COX) subunit 5B protein was decreased in ALI, although mRNA level was increased. The oxidation of ferrocytochrome C to ferricytochrome C was used for detection of cytochrome C oxidase activity of isolated mitochondria and resulted in a trend of higher activity in ALI. Furthermore, quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. To evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenized cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In contrast, blocking with 2-desoxy-D-glucose (2DG) significantly reduced ATP content in ALI and SMC. These results indicate a metabolic shift in IPEC-1 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis.
似乎提高空气-液界面（ALI）中小肠肠上皮细胞的氧气供应是导致其形态、代谢和功能变化的主要原因。猪小肠上皮细胞 1（IPEC-1）的研究较少，很少被用作肠道屏障模型，但在 ALI 培养过程中其细胞形态发生了显著改变。我们旨在回答以下问题：观察到的形态变化是否伴随着代谢功能的改变？对淹没培养（SMC）和 ALI 培养的微阵列分析鉴定出 830 个显著调节的基因。随后的功能聚类显示，代谢途径、碳代谢、糖酵解和缺氧诱导因子（HIF）信号转导中存在 31 条途径的改变，其中受调节基因数量最多。此外，作为糖酵解和氧化磷酸化之间代谢转换的介质 HIF-1α在 ALI 中表现出 mRNA 水平增加的趋势，而核内 HIF-1α含量则降低。氧化磷酸化的候选基因，如线粒体标志物，其 mRNA 水平升高，这通过 Western blot 分析得到了证实。细胞色素 C 氧化酶（COX）亚基 5B 蛋白在 ALI 中减少，尽管其 mRNA 水平增加。用分离的线粒体检测细胞色素 C 氧化酶活性时，用还原型细胞色素 C 氧化为高铁细胞色素 C 来检测，结果表明 ALI 中的活性呈上升趋势。此外，通过定量细胞培养上清液中的葡萄糖和乳酸浓度，发现 ALI 中葡萄糖水平显著降低，乳酸生成减少。为了评估能量代谢，我们在匀浆细胞悬浮液中测量了细胞三磷酸腺苷（ATP）的聚集，结果显示其水平相似。然而，在 ALI 中应用解偶联剂 FCCP 降低了 ATP 水平，但在 SMC 中没有。相反，用 2-脱氧-D-葡萄糖（2DG）阻断则显著降低了 ALI 和 SMC 中的 ATP 含量。这些结果表明，在 ALI 条件下培养的 IPEC-1 发生了代谢转变，增强了氧化磷酸化并抑制了糖酵解。
