Solini Anna, Chiozzi Paola, Morelli Anna, Passaro Angela, Fellin Renato, Di Virgilio Francesco
Department of Internal Medicine, University of Pisa, Italy.
J Cell Physiol. 2003 Dec;197(3):435-44. doi: 10.1002/jcp.10379.
Extracellular ATP is an ubiquitous mediator that regulates several cellular functions via specific P2 plasma membrane receptors (P2Rs), for which a role in modulating intracellular glucose metabolism has been recently suggested. We have investigated glucose uptake in response to P2Rs stimulation in fibroblasts from type 2 diabetic (T2D) patients and control subjects. P2Rs expression was evaluated by RT-PCR; intracellular calcium release by fluorometry; glucose transporter (GLUT1) translocation by immunoblotting and chemiluminescence; glucose uptake was measured with 2-deoxy-D-[1-(3)H]glucose (2-DOG) and ATP by luminometry. Cells from T2D patients, in contrast to those from healthy controls, showed no increase in glucose uptake after ATP stimulation; extracellular ATP caused, however, a similar GLUT1 recruitment to the plasma membrane in both groups. P2Rs expression did not differ between fibroblasts from diabetic and healthy subjects, but while plasma membrane depolarization, a P2X-mediated response was similar in both groups, no evident intracellular calcium increase was detectable in the cells from the former group. The calcium response in fibroblasts from diabetics was restored by co-incubation with apyrase or hexokinase, suggesting that P2YRs in those cells were normally expressed but chronically desensitised. In support to this finding, fibroblasts from T2D subjects secreted a two-fold larger amount of ATP compared to controls. Pre-treatment with apyrase or hexokinase also restored ATP stimulated glucose uptake in fibroblasts from diabetic subjects. These results suggest that extracellular ATP plays a role in the modulation of glucose transport via GLUT1, and that the P2Y-dependent GLUT1 activation is deficient in fibroblasts from T2D individuals. Our observations may point to additional therapeutic targets for improving glucose utilization in diabetes.
细胞外ATP是一种普遍存在的介质，它通过特定的P2质膜受体（P2Rs）调节多种细胞功能，最近有人提出其在调节细胞内葡萄糖代谢中发挥作用。我们研究了2型糖尿病（T2D）患者和对照受试者成纤维细胞中P2Rs刺激后葡萄糖摄取的情况。通过RT-PCR评估P2Rs表达；通过荧光测定法评估细胞内钙释放；通过免疫印迹和化学发光评估葡萄糖转运蛋白（GLUT1）易位；用2-脱氧-D-[1-(3)H]葡萄糖（2-DOG）和ATP通过发光测定法测量葡萄糖摄取。与健康对照者的细胞相比，T2D患者的细胞在ATP刺激后葡萄糖摄取没有增加；然而，细胞外ATP在两组中都引起了类似的GLUT1向质膜的募集。糖尿病患者和健康受试者的成纤维细胞中P2Rs表达没有差异，但虽然质膜去极化，一种P2X介导的反应在两组中相似，但在前一组细胞中未检测到明显的细胞内钙增加。糖尿病患者成纤维细胞中的钙反应通过与腺苷三磷酸双磷酸酶或己糖激酶共同孵育得以恢复，这表明这些细胞中的P2YRs正常表达但长期脱敏。支持这一发现的是，T2D受试者的成纤维细胞分泌的ATP量是对照组的两倍。用腺苷三磷酸双磷酸酶或己糖激酶预处理也恢复了糖尿病受试者成纤维细胞中ATP刺激的葡萄糖摄取。这些结果表明，细胞外ATP在通过GLUT1调节葡萄糖转运中发挥作用，并且T2D个体的成纤维细胞中P2Y依赖性GLUT1激活存在缺陷。我们的观察结果可能为改善糖尿病患者葡萄糖利用指出额外的治疗靶点。
