The classical methods for determining glucose uptake rates in living cells involve the use of isotopically labeled 2-deoxy-d-glucose or 3-O-methyl-d-glucose, which enter cells via well-characterized membrane transporters of the SLC2A and SLC5A families, respectively. These classical methods, however, are increasingly being displaced by high-throughput assays that utilize fluorescent analogs of glucose. Among the most commonly used of these analogs are 2-NBDG and 6-NBDG, which contain a bulky 7-nitro-2,1,3-benzoxadiazol-4-yl-amino moiety in place of a hydroxy group on d-glucose. This fluorescent group significantly alters both the size and shape of these molecules compared to glucose, calling into question whether they actually enter cells by the same transport mechanisms. In this study, we took advantage of the well-defined glucose uptake mechanism of L929 murine fibroblasts, which rely exclusively on the Glut1/Slc2a1 membrane transporter. We demonstrate that neither pharmacologic inhibition of Glut1 nor genetic manipulation of its expression has a significant impact on the binding or uptake of 2-NBDG or 6-NBDG by L929 cells, though both approaches significantly impact [³H]-2-deoxyglucose uptake rates. Together these data indicate that 2-NBDG and 6-NBDG can bind and enter mammalian cells by transporter-independent mechanisms, which calls into question their utility as an accurate proxy for glucose transport.

确定活细胞中葡萄糖摄取率的经典方法包括使用同位素标记的 2-脱氧-d-葡萄糖或 3-O-甲基-d-葡萄糖，它们通过 SLC2A 和 SLC5A 家族的充分表征的膜转运蛋白进入细胞， 分别。然而，这些经典方法越来越多地被利用葡萄糖荧光类似物的高通量测定所取代。这些类似物中最常用的是 2-NBDG 和 6-NBDG，它们含有一个庞大的 7-nitro-2,1,3-benzoxadiazol-4-yl-amino 部分代替d上的一个羟基-葡萄糖。与葡萄糖相比，这种荧光基团显着改变了这些分子的大小和形状，让人质疑它们是否真的通过相同的运输机制进入细胞。在这项研究中，我们利用了 L929 小鼠成纤维细胞明确定义的葡萄糖摄取机制，该机制完全依赖于 Glut1/Slc2a1 膜转运蛋白。我们证明 Glut1 的药理学抑制和其表达的基因操作都不会对 L929 细胞对 2-NBDG 或 6-NBDG 的结合或摄取产生显着影响，尽管这两种方法都会显着影响 [ ³H]-2-脱氧葡萄糖摄取率。这些数据共同表明 2-NBDG 和 6-NBDG 可以通过独立于转运蛋白的机制结合并进入哺乳动物细胞，这对它们作为葡萄糖转运准确代理的效用提出了质疑。