Rivera Diaz Paola A, Gómez Camargo Doris E, Ondo-Méndez Alejandro, Gómez-Alegría Claudio J
Universidad Nacional de Colombia, Sede Bogotá, Facultad de Ciencias, Departamento de Farmacia, Grupo de investigación UNIMOL, Av. Carrera 30 #45-03, Bogotá, Código Postal 111321, Colombia.
Universidad de Cartagena, Facultad de Medicina, Doctorado en Medicina Tropical del SUE Caribe, Grupo UNIMOL, Cartagena, Colombia.
Heliyon. 2020 Feb 27;6(2):e03422. doi: 10.1016/j.heliyon.2020.e03422. eCollection 2020 Feb.
The quantitation of glucose consumption in animal cell cultures is mainly based on the use of radiolabeled or fluorescent analogues, resulting in expensive and tedious procedures, requiring special equipment and, sometimes, with potential health and environmental risks.
The objective of this work was to evaluate the application of a blood plasma colorimetric assay to quantify glucose consumption in cultures of adipose cells.
We worked with 3T3-L1 adipose cells differentiated by 7-8 days, which were exposed to different initial glucose concentrations (5.5, 2.8 and 1.4 mM) for variable times, either in the absence or the presence of 100 nM insulin. Using a commercial colorimetric glucose assay, extracellular glucose was determined, and glucose uptake was calculated as the difference between the initial and final glucose concentration.
The colorimetric assay allowed us to quantify glucose uptake in our cell model, observing a linear response over time ( ≥0.9303) to the different glucose concentrations, both in the basal and insulin-induced condition. The insulin-stimulated glucose consumption was higher than basal consumption at all glucose concentrations evaluated, but significant differences were observed at 120-, 360- and 480-min in glucose 5.5 mM (p ≤ 0.01, n = 5), and 240 min in glucose 1.4 mM (p ≤ 0.01, n = 5). A of 4.1 and 5.9 nmol/ml/min (basal and insulin-induced, respectively) and a of 1.1 mM (same in basal vs insulin-stimulated) were calculated. The bioassay was also useful in a pharmacological context: in glucose 1.4 mM, glucose consumption showed an effect that depended on insulin concentration, with a calculated EC of 18.4 ± 1.1 nM.
A simple and low-cost bioassay is proposed to quantify glucose consumption in 3T3-L1 adipose cells.
动物细胞培养中葡萄糖消耗的定量主要基于使用放射性标记或荧光类似物，这导致程序昂贵且繁琐，需要特殊设备，并且有时存在潜在的健康和环境风险。
本研究的目的是评估血浆比色法在定量脂肪细胞培养中葡萄糖消耗的应用。
我们使用分化7-8天的3T3-L1脂肪细胞，在不存在或存在100 nM胰岛素的情况下，将其暴露于不同的初始葡萄糖浓度（5.5、2.8和1.4 mM）可变时间。使用商业比色葡萄糖测定法测定细胞外葡萄糖，并将葡萄糖摄取量计算为初始和最终葡萄糖浓度之间的差值。
比色测定法使我们能够在细胞模型中定量葡萄糖摄取，在基础和胰岛素诱导条件下，观察到对不同葡萄糖浓度随时间呈线性反应（r≥0.9303）。在所有评估的葡萄糖浓度下，胰岛素刺激的葡萄糖消耗均高于基础消耗，但在5.5 mM葡萄糖浓度下，120、360和480分钟时观察到显著差异（p≤0.01，n = 5），在1.4 mM葡萄糖浓度下，240分钟时观察到显著差异（p≤0.01，n = 5）。计算出基础和胰岛素诱导条件下的Vmax分别为4.1和5.9 nmol/ml/min，Km为1.1 mM（基础与胰岛素刺激条件下相同）。该生物测定法在药理学背景下也很有用：在1.4 mM葡萄糖浓度下，葡萄糖消耗显示出依赖于胰岛素浓度的效应，计算出的EC50为18.4±1.1 nM。
提出了一种简单且低成本的生物测定法来定量3T3-L1脂肪细胞中的葡萄糖消耗。
