kn-62 and Diabetes-Mellitus--Type-2

We have investigated expression and function of the P2X7 receptor in fibroblasts from healthy subjects and patients with type 2 diabetes.. Fibroblasts were isolated from skin biopsies. P2X7 receptor expression in both cell populations was measured by functional assays, RT-PCR, fluorescence-activated cell sorter, and immunoblotting. We found that fibroblasts from diabetic subjects are characterized by enhanced P2X7-mediated responses as indicated by increased shape changes, microvesiculation, enhanced fibronectin and interleukin 6 secretion, and accelerated apoptosis. These responses were blocked by preincubation with the P2X blockers KN-62, oxidized ATP, or pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid). Furthermore, we also found a higher level of spontaneous fibronectin secretion and of apoptosis in fibroblasts from diabetic compared with healthy subjects. Both higher basal level of fibronectin secretion and spontaneous rate of apoptosis were likely attributable to the increased pericellular concentration of ATP because fibroblasts from diabetic subjects released 3x as much ATP into the supernatants compared with fibroblasts from healthy subjects.. We conclude that fibroblasts from type 2 diabetes patients are characterized by a hyperactive purinergic loop based either on a higher level of ATP release or on increased P2X7 reactivity.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Diphosphate; Adenosine Triphosphate; Apoptosis; Apyrase; Autocrine Communication; Cell Shape; Cytidine Triphosphate; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Fibroblasts; Fibronectins; Gene Expression Regulation; Humans; Interleukin-6; Membrane Potentials; Paracrine Communication; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Uridine Diphosphate; Uridine Triphosphate

Stimulation of AMP-activated protein kinase (AMPK) in skeletal muscle and liver is seen as an exciting prospect for the treatment of type 2 diabetes. However, we have recently demonstrated that changes in AMPK activity accompany the exposure of pancreatic islet beta-cells to elevated glucose concentrations and may be involved in the activation of insulin secretion. Here, we discuss this hypothesis and explore the potential role of changes in AMPK activity in the actions of other secretagogues. Amino acids decreased AMPK activity in MIN6 beta-cells with an order of potency for inhibition: arg=leu < gln= leu + glu < glucose, which was closely correlated with the stimulation of insulin release (r2=0.76). By contrast, increases in intracellular Ca2+ concentration provoked by cell depolarization with KCl activated AMPK in the face of increased free intracellular ATP concentrations. Elevation of intracellular cAMP levels with isobutylmethylxanthine or forskolin had no effect on AMPK activity. We conclude that metabolizable amino acids regulate AMPK in the beta-cell via increases in the cytosolic ATP/AMP ratio and via phosphorylation by the upstream kinase LKB1. Intracellular Ca2+ ions may activate AMPK by calmodulin kinase 1 kinase-mediated phosphorylation. The latter may act as a novel feedback mechanism to inhibit excessive insulin secretion under some circumstances.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amino Acids; AMP-Activated Protein Kinases; Animals; Calcium; Cell Line, Tumor; Diabetes Mellitus, Type 2; Enzyme Inhibitors; Insulinoma; Islets of Langerhans; Kinetics; Mice; Multienzyme Complexes; Protein Serine-Threonine Kinases