AbstractExtracellular 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‐[1‐3H]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. J. Cell. Physiol. 197: 435–444, 2003© 2003 Wiley‐Liss, Inc.