Background and Aims: PlY receptors (p2YR) are membrane-spanning G-proteincoupled receptors whose activation by extracellular ATP (eATP) triggers generation ofinositoll,4,5-triphosphate and calcium mobilization from intracellular stores. They are involved in regulating platelet aggregation, cytokine release, smooth muscle cells contraction. Recently, a role of PZYR in modulating glucose transport (GT) in rat cardiomyocytes has been suggested. Aim of this study was to evaluate the effect 01 P2YR stimulation on glucose uptake (GU) in skin fibroblasts from 6 healthy subjects (C) and 6 type 2 diabetic patients (T2D). Materials and Methods: PZYR expression was evaluated by RT-PCR; changes in intracellular calcium concentration by the fluorescent indicator Fura21A.\I; GLUTl was identified in three membrane fractions by immunoblotting and chemiluminescence; GU was measured with the analogue 2deoxy- D-[J-3H] glucose (2-DOG) and eATP in the medium by Iuminornetry with the luciferin-Iuciferase assay. Results: 2-DOG uptake from T2D was basically insensitive to eATP stimulation (basallOO±l2 in C and 85±15 pmol/mg/min in T2D; with ATP I 01.\1230±21 in C and 126±13 pmol/mg/min in T2D). GLUTI was equally expressed in C and T2D; its content in the Golgi, however, was much lower in T2D. eATP was able to promote GLUTI association to the plasma membrane in both cells, but this event pcr se was not sufficient to drive an increased GU in T2D. Expression ofP2YR did not differ in the two groups. Intracellular calcium release, a functional response tipically triggered by P2YR, was reduced in T2D respect to C (102 vs 248 0.\1). Pretreatment with hexokinase, an ATP hydrolysing enzyme, completely restored P2YR-mediated calcium release in TID (228 vs 217 nM in C), suggesting a desensitized state of this receptors in T2D. This hypothesis was confirmed by a threefold higher ATP content in the supernatants of T2D compared to C (0.2±O.5 and 0.08±O.2 micrograms ATP/1.ooo.ooo cells). Intracellular ATP, otherwise, did not differ between the two cell populations. Accordingly, incubation in the presence of hexokinase re-established ATP-dependent GU (140±1O in C and 156±12 pmol/mg/min in TID). Condusions: extracellular ATP appears to modulate insulinindependent GT, PZYR-dependent GLUTI activation being deficient in type 2 diabetes. These
Defective P2Y purinergic receptor function induces impaired glucose transport in type 2 diabetes.
CHIOZZI, Paola;PASSARO, Angelina;FELLIN, Renato;Di Virgilio, F.
2001
Abstract
Background and Aims: PlY receptors (p2YR) are membrane-spanning G-proteincoupled receptors whose activation by extracellular ATP (eATP) triggers generation ofinositoll,4,5-triphosphate and calcium mobilization from intracellular stores. They are involved in regulating platelet aggregation, cytokine release, smooth muscle cells contraction. Recently, a role of PZYR in modulating glucose transport (GT) in rat cardiomyocytes has been suggested. Aim of this study was to evaluate the effect 01 P2YR stimulation on glucose uptake (GU) in skin fibroblasts from 6 healthy subjects (C) and 6 type 2 diabetic patients (T2D). Materials and Methods: PZYR expression was evaluated by RT-PCR; changes in intracellular calcium concentration by the fluorescent indicator Fura21A.\I; GLUTl was identified in three membrane fractions by immunoblotting and chemiluminescence; GU was measured with the analogue 2deoxy- D-[J-3H] glucose (2-DOG) and eATP in the medium by Iuminornetry with the luciferin-Iuciferase assay. Results: 2-DOG uptake from T2D was basically insensitive to eATP stimulation (basallOO±l2 in C and 85±15 pmol/mg/min in T2D; with ATP I 01.\1230±21 in C and 126±13 pmol/mg/min in T2D). GLUTI was equally expressed in C and T2D; its content in the Golgi, however, was much lower in T2D. eATP was able to promote GLUTI association to the plasma membrane in both cells, but this event pcr se was not sufficient to drive an increased GU in T2D. Expression ofP2YR did not differ in the two groups. Intracellular calcium release, a functional response tipically triggered by P2YR, was reduced in T2D respect to C (102 vs 248 0.\1). Pretreatment with hexokinase, an ATP hydrolysing enzyme, completely restored P2YR-mediated calcium release in TID (228 vs 217 nM in C), suggesting a desensitized state of this receptors in T2D. This hypothesis was confirmed by a threefold higher ATP content in the supernatants of T2D compared to C (0.2±O.5 and 0.08±O.2 micrograms ATP/1.ooo.ooo cells). Intracellular ATP, otherwise, did not differ between the two cell populations. Accordingly, incubation in the presence of hexokinase re-established ATP-dependent GU (140±1O in C and 156±12 pmol/mg/min in TID). Condusions: extracellular ATP appears to modulate insulinindependent GT, PZYR-dependent GLUTI activation being deficient in type 2 diabetes. TheseI documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
