Glucose consumption of vascular cell types in culture: toward optimization of experimental conditions
Glucose consumption of vascular cell types in culture: toward optimization of experimental conditions
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DOI：
10.1152/ajpcell.00257.2021
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发表时间：
2022-01-01
影响因子：
5.5
通讯作者：
Eguchi, Satoru
中科院分区：
文献类型：
--
作者：
Torimoto, Keiichi;Okuno, Keisuke;Eguchi, Satoru
在这项研究中，我们已经寻找了一个最佳的培养基葡萄糖浓度和比较葡萄糖消耗在三种血管细胞类型，内皮细胞（EC），血管平滑肌细胞（VSMCs），和血管外膜成纤维细胞（AF）有或无血管紧张素II（AngII）刺激。在具有标准低（100 mg/dL）、标准高（450 mg/dL）或混合中等（275 mg/dL）葡萄糖浓度的1 mL DMEM中进行的近汇合6孔实验中，在所有细胞类型中观察到稳定且显著的葡萄糖消耗。孵育48小时后，含有低葡萄糖的培养基减少至几乎0 mg/dL，含有高葡萄糖的培养基保持在约275 mg/dL的显著较高水平，含有中等葡萄糖的培养基保持更接近生理范围。AngII处理增强了AF和VSMC的葡萄糖消耗，但在EC中没有。在AF中也观察到AngII增强的细胞外酸化速率。在AF中，AngII在48 h对靶蛋白的诱导取决于所用的葡萄糖浓度。在低葡萄糖培养基中，葡萄糖调节蛋白78或己糖激酶II的诱导最高，而VCAM-1的诱导最低。特异性抑制剂的使用进一步表明血管紧张素II 1型受体和糖酵解在AngII诱导的成纤维细胞活化中的重要作用。总体而言，本研究表明，当标准低或高葡萄糖培养基与血管细胞一起使用时，低血糖或高血糖状况的风险较高。此外，这些条件可能会显着改变实验结果。在任何培养实验中应监测培养基葡萄糖浓度，建议所有血管细胞类型使用中等葡萄糖培养基。
In this study, we have looked for an optimum media glucose concentration and compared glucose consumption in three vascular cell types, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and adventitial fibroblasts (AFs) with or without angiotensin II (AngII) stimulation. In a subconfluent 6-well experiment in 1 mL DMEM with a standard low (100 mg/dL), a standard high (450 mg/dL), or a mixed middle (275 mg/dL) glucose concentration, steady and significant glucose consumption was observed in all cell types. After 48-h incubation, media that contained low glucose was reduced to almost 0 mg/dL, media that contained high glucose remained significantly higher at -275 mg/dL, and media that contained middle glucose remained closer to physiological range. AngII treatment enhanced glucose consumption in AFs and VSMCs but not in ECs. Enhanced extracellular acidification rate by AngII was also observed in AFs. In AFs, AngII induction of target proteins at 48 h varied depending on the glucose concentration used. In low glucose media, induction of glucose regulatory protein 78 or hexokinase II was highest, whereas induction of VCAM-1 was lowest. Utilization of specific inhibitors further suggests essential roles of angiotensin II type-1 receptor and glycolysis in AngII-induced fibroblast activation. Overall, this study demonstrates a high risk of hypo- or hyperglycemic conditions when standard low or high glucose media is used with vascular cells. Moreover, these conditions may significantly alter experimental outcomes. Media glucose concentration should be monitored during any culture experiments and utilization of middle glucose media is recommended for all vascular cell types.