RESUMO
We investigated the impact of a diabetic state with hyperglycemia on morphometry of ß cell mitochondria and modifying influence of a K (+) -ATP channel opener and we related in vivo findings with glucose effects in vitro. For in vivo experiments islets from syngeneic rats were transplanted under the kidney capsule to neonatally streptozotocin-diabetic or non-diabetic recipients. Diabetic recipients received vehicle, or tifenazoxide (NN414), intragastrically for 9 weeks. Non-diabetic rats received vehicle. Transplants were excised 7 d after cessation of treatment (wash-out) and prepared for electron microscopy. Morphological parameters were measured from approx. 25,000 mitochondria. Rat islets were cultured in vitro for 2-3 weeks at 27 or 11 (control) mmol/l glucose. Transplants to diabetic rats displayed decreased numbers of mitochondria (-31%, p < 0.05), increased mitochondrial volume and increased mitochondrial outer surface area, p < 0.001. Diabetes increased variability in mitochondrial size with frequent appearance of mega-mitochondria. Tifenazoxide partly normalized diabetes-induced effects, and mega-mitochondria disappeared. Long-term culture of islets at 27 mmol/l glucose reproduced the in vivo morphological abnormalities. High-glucose culture was also associated with reduced ATP and ADP contents, reduced oxygen consumption, reduced signaling by MitoTracker Red and reduction of mitochondrial proteins (complexes I-IV), OPA 1 and glucose-induced insulin release. We conclude that (1) a long-term diabetic state leads to a reduced number of mitochondria and to distinct morphological abnormalities which are replicated by high glucose in vitro; (2) the morphological abnormalities are coupled to dysfunction; (3) K (+) -ATP channel openers may have potential to partly reverse glucose-induced effects.
Assuntos
Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/patologia , Hiperglicemia/patologia , Ilhotas Pancreáticas/patologia , Mitocôndrias/patologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Óxidos S-Cíclicos/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Hiperglicemia/metabolismo , Hipoglicemiantes/farmacologia , Ilhotas Pancreáticas/metabolismo , Masculino , Microscopia Eletrônica , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Ratos , Ratos Endogâmicos WF , Ratos Sprague-DawleyRESUMO
2-(4-Methoxyphenoxy)-5-nitro-N-(4-sulfamoylphenyl)benzamide and close analogues inhibit glucose stimulated insulin release through activation of Kir6.2/SUR1 K(ATP) channels of beta cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Benzamidas/química , Benzamidas/farmacologia , Diazóxido/análogos & derivados , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio/metabolismo , Receptores de Droga/metabolismo , Transportadores de Cassetes de Ligação de ATP/agonistas , Trifosfato de Adenosina/metabolismo , Animais , Benzotiadiazinas/química , Benzotiadiazinas/farmacologia , Diazóxido/química , Diazóxido/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Canais de Potássio/agonistas , Canais de Potássio Corretores do Fluxo de Internalização/agonistas , Piridinas/química , Piridinas/farmacologia , Ratos , Receptores de Droga/agonistas , Receptores de Sulfonilureias , Tiadiazinas/química , Tiadiazinas/farmacologiaRESUMO
ATP-sensitive K(+) (K(ATP)) channels are activated by a diverse group of compounds known as potassium channel openers (PCOs). Here, we report functional studies of the Kir6.2/SUR1 Selective PCO 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide (NNC 55-9216). We recorded cloned K(ATP) channel currents from inside-out patches excised from Xenopus laevis oocytes heterologously expressing Kir6.2/SUR1, Kir6.2/SUR2A, or Kir6.2/SUR2B, corresponding to the beta-cell, cardiac, and smooth muscle types of the K(ATP) channel. NNC 55-9216 reversibly activated Kir6.2/SUR1 currents (EC(50) = 16 micromol/l). This activation was dependent on intracellular MgATP and was abolished by mutation of a single residue in the Walker A motifs of either nucleotide-binding domain of SUR1. The drug had no effect on Kir6.2/SUR2A or Kir6.2/SUR2B currents. We therefore used chimeras of SUR1 and SUR2A to identify regions of SUR1 involved in the response to NNC 55-9216. Activation was completely abolished and significantly reduced by swapping transmembrane domains 8-11. The reverse chimera consisting of SUR2A with transmembrane domains 8-11 and NBD2 consisting SUR1 was activated by NNC 55-9216, indicating that these SUR1 regions are important for drug activation. [(3)H]glibenclamide binding to membranes from HEK293 cells transfected with SUR1 was displaced by NNC 55-9216 (IC(50) = 105 micromol/l), and this effect was impaired when NBD2 of SUR1 was replaced by that of SUR2A. These results suggest NNC 55-9216 is a SUR1-selective PCO that requires structural determinants, which differ from those needed for activation of the K(ATP) channel by pinacidil and cromakalim. The high selectivity of NNC 55-9216 may prove to be useful for studies of the molecular mechanism of PCO action.