Abstract
BackgroundMutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene lead to the disease cystic fibrosis (CF). Although patients with CF often have disturbances in glucose metabolism including impaired insulin release, no previous studies have tested the hypothesis that CFTR has a biological function in pancreatic beta-cells.MethodsExperiments were performed on islets and single beta-cells from human donors and NMRI-mice. Detection of CFTR was investigated using PCR and confocal microscopy. Effects on insulin secretion were measured with radioimmunoassay (RIA). The patch-clamp technique was used to measure ion channel currents and calcium-dependent exocytosis (as changes in membrane capacitance) on single cells with high temporal resolution. Analysis of ultrastructure was done on transmission electron microscopy (TEM) images.ResultsWe detected the presence of CFTR and measured a small CFTR conductance in both human and mouse beta-cells. The augmentation of insulin secretion at 16.7 mM glucose by activation of CFTR by cAMP (forskolin (FSK) or GLP-1) was significantly inhibited when CFTR antagonists (GlyH-101 and/or CFTRinh-172) were added. Likewise, capacitance measurements demonstrated reduced cAMP-dependent exocytosis upon CFTR-inhibition, concomitant with a decreased number of docked insulin granules. Finally, our studies demonstrate that CFTR act upstream of the chloride channel Anoctamin 1 (ANO1; TMEM16A) in the regulation of cAMP- and glucose-stimulated insulin secretion.ConclusionOur work demonstrates a novel function for CFTR as a regulator of pancreatic beta-cell insulin secretion and exocytosis, and put forward a role for CFTR as regulator of ANO1 and downstream priming of insulin granules prior to fusion and release of insulin. The pronounced regulatory effect of CFTR on insulin secretion is consistent with impaired insulin secretion in patients with CF.
Highlights
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene lead to the disease cystic fibrosis (CF)
Cystic fibrosis transmembrane conductance regulator (CFTR)-antagonists inhibit insulin secretion and an ATP-sensitive and cAMP-dependent current in mouse and human beta-cells To investigate if CFTR affects beta-cell function, the influence of CFTR on insulin secretion was measured from isolated human and mouse pancreatic islets subjected to glucose-induced insulin secretion assays
CFTRinh-172, another antagonist against CFTR, decreased both FSK and GLP-1 enhanced insulin secretion (Figures 1C, D). Both inhibitors are specific for CFTR, and whereas CFTRinh-172 binds to the intracellular domain of CFTR [23] and thereby closes the channel, GlyH-101 is an open-channel blocker [24]
Summary
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene lead to the disease cystic fibrosis (CF). The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel that belongs to the family of ATP-binding cassette (ABC)-transporters [1]. As many other ABC-transporters, CFTR contains two membrane spanning domains (MSDs) and two nucleotide binding domains (NBDs) that interact with ATP. CFTR is primarily present in epithelial cells in airways, intestine and in cells with exocrine functions. Mutations in the gene encoding the channel protein complex (cftr) cause the autosomal recessive disease cystic fibrosis (CF). In patients with CF the defective chloride transport through CFTR leads to production of thick viscous mucus caused by a disturbed ion and water transport across epithelial membranes. The aberrant secretory functions cause obstruction of the distal airways and intestine, pancreatitis and malabsorption [3]
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