Defective exocytosis and processing of insulin in a cystic fibrosis mouse model

Abstract

Cystic fibrosis-related diabetes (CFRD) is a common complication for patients with cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The cause of CFRD is unclear, but a commonly observed reduction in first-phase insulin secretion suggests defects at the beta cell level. Here we aimed to examine alpha and beta cell function in the Cftr tm1 EUR/F508del mouse model (C57BL/6J), which carries the most common human mutation in CFTR, the F508del mutation. CFTR expression, beta cell mass, insulin granule distribution, hormone secretion and single cell capacitance changes were evaluated using islets (or beta cells) from F508del mice and age-matched wild type (WT) mice aged 7–10 weeks. Granular pH was measured with DND-189 fluorescence. Serum glucose, insulin and glucagon levels were measured in vivo, and glucose tolerance was assessed using IPGTT. We show increased secretion of proinsulin and concomitant reduced secretion of C-peptide in islets from F508del mice compared to WT mice. Exocytosis and number of docked granules was reduced. We confirmed reduced granular pH by CFTR stimulation. We detected decreased pancreatic beta cell area, but unchanged beta cell number. Moreover, the F508del mutation caused failure to suppress glucagon secretion leading to hyperglucagonemia. In conclusion, F508del mice have beta cell defects resulting in (1) reduced number of docked insulin granules and reduced exocytosis and (2) potential defective proinsulin cleavage and secretion of immature insulin. These observations provide insight into the functional role of CFTR in pancreatic islets and contribute to increased understanding of the pathogenesis of CFRD.

Short-term CFTR inhibition reduces islet area in C57BL/6 mice

“…When the number of insulin, or glucagon-positive cells were counted as a percentage of total islet cells in this model of short-term CFTR inhibition, it was evident that the reduction in islet size resulted from smaller islet cells rather than loss of cells. This is consistent with recent observations in the ΔF508 mouse 19 and raises the attractive possibility that early interventions to prevent reductions in islet mass may assist in the maintenance of normal islet size and function in CF. In line with the reductions in islet size, insulin content and peripheral insulin concentrations were also significantly reduced.…”

“…The pancreatic and intestinal phenotypes of CFTR-inhibited mice were largely consistent with CF disease. Islet size was reduced in CFTR-inhibited animals, and the ΔF508 and CFTR KO animals, consistent with the literature 17,19 . In addition, ductal dilatation, a hallmark of CF disease, was evident in all three mouse models studied here.…”

“…Reductions in insulin secretion in the face of glucose challenge have been reported in several CF animal 15,16,19,28 and cell models 5–9 , and in patients with CF 28–33 . Fontes et al .…”

“…In this model, a significant increase in the number of small islets was observed in neonates 15 . Subsequent studies have repeatedly shown reductions in islet mass or beta cell area in animal models of CF 16–19 and in post mortem pancreas from patients with CF 10,20 including children under the age of 4 years 20 . In the latter study, beta cell area was reduced by as much as 50% in children less than 4 years old when compared with age-matched controls and this was shown to be independent of the degree of exocrine pancreatic fibrosis or the presence/absence of diabetes 20 .…”

Chloride transporters and channels in β-cell physiology: revisiting a 40-year-old model

“…Importantly and contrary to general awareness, Cftr does not need to be an abundant PM-confined Cl – channel to have a function; Cftr is active in the trans-Golgi network [113], endosomes and lysosomes [114] and in acidic organelles/granules [115]. Moreover, it has been suggested that Cftr regulates exocytosis in β-cells [107], a hypothesis recently tested and extended to a potential role of Cftr in insulin processing [116], a phenomenon of the acidified insulin granule [117]. Indeed, exocytosis events and the number of insulin-containing granules docked at the PM were reduced in β-cells lacking functional Cftr , whereas stimulation of Cftr increased granular acidification [116].…”