RESUMEN
Apoptosis of pancreatic beta-cells is an important factor in the pathophysiology of diabetes. Previously, we have shown that the "phytoestrogen" resveratrol can induce beta-cell apoptosis dependent on the expression of sulfonylurea receptor (SUR) 1, the regulatory subunit of pancreatic ATP-sensitive K(+) channels. Here, we investigate whether 17beta-estradiol also influences beta-cell apoptosis in a SUR1-dependent manner. Therefore, islets from wild type or SUR1 knock-out mice, clonal beta-cells, or HEK293 cells expressing different SUR forms were treated with 17beta-estradiol or estrone. Different apoptotic parameters were determined and estrogen binding to SUR was analyzed. In murine islets, 17beta-estradiol treatment resulted in significant apoptotic changes, which in their nature (either apoptotic or anti-apoptotic) were dependent on the age of the animal. These effects were not observed in SUR1 knock-out mice. Furthermore, 17beta-estradiol, which specifically binds to SUR, induced enhanced apoptosis in SUR1-expressing HEK293 cells and clonal beta-cells, whereas apoptosis in recombinant cells expressing SUR2A or SUR2B (cardiac or vascular SUR-isoforms) or sham-transfected control cells was significantly lower. The apoptotic potency of 17beta-estradiol was much higher than that of resveratrol or estrone. SUR1-specific 17beta-estradiol-induced apoptosis was either abolished by the mutation M1289T in transmembrane helix 17 of SUR1 or clearly enhanced by two mutations in nucleotide binding fold 2 (R1379C, R1379L). In conclusion, 17beta-estradiol treatment modulates beta-cell apoptosis under specific involvement of SUR1 in an age-dependent manner. 17beta-Estradiol-induced apoptosis can be influenced by certain SUR1 mutations. These findings may contribute to the understanding of pathophysiological changes in beta-cell mass and could, for instance, provide interesting aspects concerning the etiology of gestational diabetes.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Apoptosis/efectos de los fármacos , Diabetes Gestacional/metabolismo , Estradiol/farmacología , Estrógenos/farmacología , Células Secretoras de Insulina/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Receptores de Droga/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Animales , Apoptosis/genética , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Diabetes Gestacional/genética , Diabetes Gestacional/patología , Estrona/farmacología , Femenino , Humanos , Células Secretoras de Insulina/patología , Masculino , Ratones , Ratones Noqueados , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/genética , Canales de Potasio/genética , Canales de Potasio/metabolismo , Canales de Potasio de Rectificación Interna/genética , Embarazo , Pliegue de Proteína , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Receptores de Droga/genética , Receptores de SulfonilureasRESUMEN
Sulfonylurea receptors (SURs) constitute the regulatory subunits of ATP-sensitive K+ channels (K(ATP) channels). SUR binds nucleotides and synthetic K(ATP) channel modulators, e.g. the antidiabetic sulfonylurea glibenclamide, which acts as a channel blocker. However, knowledge about naturally occurring ligands of SUR is very limited. In this study, we show that the plant phenolic compound trans-resveratrol can bind to SUR and displace binding of glibenclamide. Electrophysiological measurements revealed that resveratrol is a blocker of pancreatic SUR1/K(IR)6.2 K(ATP) channels. We further demonstrate that, like glibenclamide, resveratrol induces enhanced apoptosis. This was shown by analyzing different apoptotic parameters (cell detachment, nuclear condensation and fragmentation, and activities of different caspase enzymes). The observed apoptotic effect was specific to cells expressing the SUR1 isoform and was not mediated by the electrical activity of K(ATP) channels, as it was observed in human embryonic kidney 293 cells expressing SUR1 alone. Enhanced susceptibility to resveratrol was not observed in pancreatic beta-cells from SUR1 knock-out mice or in cells expressing the isoform SUR2A or SUR2B or the mutant SUR1(M1289T). Resveratrol was much more potent than glibenclamide in inducing SUR1-specific apoptosis. Treatment with etoposide, a classical inducer of apoptosis, did not result in SUR isoform-specific apoptosis. In conclusion, resveratrol is a natural SUR ligand that can induce apoptosis in a SUR isoform-specific manner. Considering the tissue-specific expression patterns of SUR isoforms and the possible effects of SUR mutations on susceptibility to apoptosis, these observations could be important for diabetes and/or cancer research.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Apoptosis/fisiología , Canales de Potasio de Rectificación Interna/fisiología , Canales de Potasio/fisiología , Receptores de Droga/fisiología , Estilbenos/farmacología , Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/genética , Animales , Apoptosis/efectos de los fármacos , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Adhesión Celular/efectos de los fármacos , Línea Celular , Etopósido/farmacología , Femenino , Humanos , Hipoglucemiantes/farmacología , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/fisiología , Riñón , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Canales de Potasio/deficiencia , Canales de Potasio/efectos de los fármacos , Canales de Potasio/genética , Canales de Potasio de Rectificación Interna/deficiencia , Canales de Potasio de Rectificación Interna/efectos de los fármacos , Canales de Potasio de Rectificación Interna/genética , Receptores de Droga/deficiencia , Receptores de Droga/efectos de los fármacos , Receptores de Droga/genética , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Resveratrol , Estilbenos/farmacocinética , Receptores de Sulfonilureas , TransfecciónRESUMEN
Sulfonylurea receptor 1 (SUR1) is the regulatory subunit of the pancreatic ATP-sensitive K+ channel (K(ATP) channel), which is essential for triggering insulin secretion via membrane depolarization. Sulfonylureas, such as glibenclamide and tolbutamide, act as K(ATP) channel blockers and are widely used in diabetes treatment. These antidiabetic substances are known to induce apoptosis in pancreatic beta-cells or beta-cell lines under certain conditions. However, the precise molecular mechanisms of this sulfonylurea-induced apoptosis are still unidentified. To investigate the role of SUR in apoptosis induction, we tested the effect of glibenclamide on recombinant human embryonic kidney 293 cells expressing either SUR1, the smooth muscular isoform SUR2B, or the mutant SUR1(M1289T) at which a single amino acid in transmembrane helix 17 (TM17) was exchanged by the corresponding amino acid of SUR2. By analyzing cell detachment, nuclear condensation, DNA fragmentation, and caspase-3-like activity, we observed a SUR1-specific enhancement of glibenclamide-induced apoptosis that was not seen in SUR2B, SUR1(M1289T), or control cells. Coexpression with the pore-forming Kir6.2 subunit did not significantly alter the apoptotic effect of glibenclamide on SUR1 cells. In conclusion, expression of SUR1, but not of SUR2B or SUR1(M1289T), renders cells more susceptible to glibenclamide-induced apoptosis. Therefore, SUR1 as a pancreatic protein could be involved in specific variation of beta-cell mass and might also contribute to the regulation of insulin secretion at this level. According to our results, TM17 is essentially involved in SUR1-mediated apoptosis. This effect does not require the presence of functional Kir6.2-containing K(ATP) channels, which points to additional, so far unknown functions of SUR.