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Background and Aims: PlY receptors (p2YR) are membrane-spanning G-proteincoupled\udreceptors whose activation by extracellular ATP (eATP) triggers generation\udofinositoll,4,5-triphosphate and calcium mobilization from intracellular stores. They\udare involved in regulating platelet aggregation, cytokine release, smooth muscle cells\udcontraction. Recently, a role of PZYR in modulating glucose transport (GT) in rat\udcardiomyocytes has been suggested. Aim of this study was to evaluate the effect 01\udP2YR stimulation on glucose uptake (GU) in skin fibroblasts from 6 healthy subjects\ud(C) and 6 type 2 diabetic patients (T2D). Materials and Methods: PZYR expression\udwas evaluated by RT-PCR; changes in intracellular calcium concentration by the\udfluorescent indicator Fura21A.\I; GLUTl was identified in three membrane fractions\udby immunoblotting and chemiluminescence; GU was measured with the analogue 2deoxy-\udD-[J-3H] glucose (2-DOG) and eATP in the medium by Iuminornetry with the\udluciferin-Iuciferase assay. Results: 2-DOG uptake from T2D was basically insensitive\udto eATP stimulation (basallOO±l2 in C and 85±15 pmol/mg/min in T2D; with ATP I\ud01.\1230±21 in C and 126±13 pmol/mg/min in T2D). GLUTI was equally expressed\udin C and T2D; its content in the Golgi, however, was much lower in T2D. eATP was\udable to promote GLUTI association to the plasma membrane in both cells, but this\udevent pcr se was not sufficient to drive an increased GU in T2D. Expression ofP2YR\uddid not differ in the two groups. Intracellular calcium release, a functional response\udtipically triggered by P2YR, was reduced in T2D respect to C (102 vs 248 0.\1).\udPretreatment with hexokinase, an ATP hydrolysing enzyme, completely restored\udP2YR-mediated calcium release in TID (228 vs 217 nM in C), suggesting a\uddesensitized state of this receptors in T2D. This hypothesis was confirmed by a threefold\udhigher ATP content in the supernatants of T2D compared to C (0.2±O.5 and\ud0.08±O.2 micrograms ATP/1.ooo.ooo cells). Intracellular ATP, otherwise, did not\uddiffer between the two cell populations. Accordingly, incubation in the presence of\udhexokinase re-established ATP-dependent GU (140±1O in C and 156±12\udpmol/mg/min in TID). Condusions: extracellular ATP appears to modulate insulinindependent\udGT, PZYR-dependent GLUTI activation being deficient in type 2\uddiabetes. Thes

Abstracts of the 37th Annual Meeting of the EASD Glasgow, United Kingdom, 9–13 September 2001