Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil.
J Membr Biol. 2011 Feb;239(3):157-65. doi: 10.1007/s00232-010-9334-6. Epub 2010 Dec 8.
The effect of glucose on the intracellular pH (pH(i)) recovery rate (dpH(i)/dt) and Na(+)-glucose transporter (SGLT) localization was investigated in HEK-293 cells, a cell line that expresses endogenous NHE1, NHE3, SGLT1, and SGLT2 proteins. The activity of the Na(+)/H(+) exchangers (NHEs) was evaluated by using fluorescence microscopy. The total and membrane protein expression levels were analyzed by immunoblotting. In cells cultivated in 5 mM glucose, the pH(i) recovery rate was 0.169 ± 0.020 (n = 6). This value did not change in response to the acute presence of glucose at 2 or 10 mM, but decreased with 25 mM glucose, an effect that was not observed with 25 mM mannitol. Conversely, the chronic effect of high glucose (25 mM) increased the pH(i) recovery rate (40%, P < 0.05), without changes in the total levels of NHE1, NHE3, or SGLT1 expression, but increasing the total cellular (50%, P < 0.05) and the plasma membrane (~100%, P < 0.01) content of SGLT2. Treatment with H-89 (10(-6) M) prevented the stimulatory effect of chronic glucose treatment on the pH(i) recovery rate and SGLT2 expression in the plasma membrane. Our results indicate that the effect of chronic treatment with a high glucose concentration is associated with increased NHEs activity and plasma membrane expression of SGLT2 in a protein kinase A-dependent way. The present results reveal mechanisms of glucotoxicity and may contribute to understanding the diabetes-induced damage of this renal epithelial cell.
我们研究了葡萄糖对 HEK-293 细胞内 pH 值（pH(i)）恢复率（dpH(i)/dt）和 Na(+)-葡萄糖转运蛋白（SGLT）定位的影响，HEK-293 细胞系表达内源性 NHE1、NHE3、SGLT1 和 SGLT2 蛋白。通过荧光显微镜评估 Na(+)/H(+)交换器（NHEs）的活性。通过免疫印迹分析总蛋白和膜蛋白表达水平。在 5mM 葡萄糖培养的细胞中，pH(i)恢复率为 0.169±0.020（n=6）。在 2 或 10mM 葡萄糖的急性存在下，该值没有变化，但在 25mM 葡萄糖时降低，而在 25mM 甘露醇时则没有观察到这种作用。相反，高葡萄糖（25mM）的慢性作用增加了 pH(i)恢复率（40%，P<0.05），但没有改变 NHE1、NHE3 或 SGLT1 的总表达水平，而是增加了 SGLT2 的总细胞（50%，P<0.05）和质膜（~100%，P<0.01）含量。用 H-89（10(-6)M）处理可防止慢性葡萄糖处理对 pH(i)恢复率和质膜中 SGLT2 表达的刺激作用。我们的结果表明，高葡萄糖浓度的慢性处理与 NHEs 活性的增加以及 SGLT2 的质膜表达有关，这是一种蛋白激酶 A 依赖性的方式。本研究结果揭示了葡萄糖毒性的机制，可能有助于理解糖尿病引起的这种肾上皮细胞损伤。
