Human and Animal Physiology, Wageningen University and Research, 6708 WD, Wageningen, The Netherlands.
Animal Nutrition Group, Wageningen University and Research, 6708 WD, Wageningen, The Netherlands.
Sci Rep. 2021 Oct 7;11(1):19961. doi: 10.1038/s41598-021-99460-0.
Intestinal epithelial cells (IECs) are crucial to maintain intestinal function and the barrier against the outside world. To support their function they rely on energy production, and failure to produce enough energy can lead to IEC malfunction and thus decrease intestinal barrier function. However, IEC metabolic function is not often used as an outcome parameter in intervention studies, perhaps because of the lack of available methods. We therefore developed a method to isolate viable IECs, suitable to faithfully measure their metabolic function by determining extracellular glycolytic and mitochondrial flux. First, various methods were assessed to obtain viable IECs. We then adapted a previously in-house generated image-analysis algorithm to quantify the amount of seeded IECs. Correcting basal respiration data of a group of piglets using this algorithm reduced the variation, showing that this algorithm allows for more accurate analysis of metabolic function. We found that delay in metabolic analysis after IEC isolation decreases their metabolic function and should therefore be prevented. The presence of antibiotics during isolation and metabolic assessment also decreased the metabolic function of IECs. Finally, we found that primary pig IECs did not respond to Oligomycin, a drug that inhibits complex V of the electron transport chain, which may be because of the presence of drug exporters. A method was established to faithfully measure extracellular glycolytic and mitochondrial flux of pig primary IECs. This tool is suitable to gain a better understanding of how interventions affect IEC metabolic function.
肠上皮细胞 (IECs) 对于维持肠道功能和防止外界入侵至关重要。为了支持其功能，它们依赖于能量产生，如果不能产生足够的能量，就会导致 IEC 功能障碍，从而降低肠道屏障功能。然而，IEC 代谢功能通常不作为干预研究的结果参数，这也许是因为缺乏可用的方法。因此，我们开发了一种分离活 IEC 的方法，通过测定细胞外糖酵解和线粒体通量来准确测量其代谢功能。首先，评估了各种方法以获得活的 IEC。然后，我们对之前内部生成的图像分析算法进行了调整，以定量接种的 IEC 数量。使用该算法校正一组仔猪的基础呼吸数据可以减少变异，表明该算法可以更准确地分析代谢功能。我们发现，在分离后延迟进行代谢分析会降低 IEC 的代谢功能，因此应加以避免。在分离和代谢评估过程中使用抗生素也会降低 IEC 的代谢功能。最后，我们发现原代猪 IEC 对抑制电子传递链复合物 V 的药物寡霉素没有反应，这可能是由于药物外排泵的存在。建立了一种方法来准确测量猪原代 IEC 的细胞外糖酵解和线粒体通量。该工具适用于更好地了解干预措施如何影响 IEC 代谢功能。
